IDENTIFYING ROLES FOR NON-ESSENTIAL GENES IN ESSENTIAL PROCESSES
by
MARC DAVID DORFMAN
A DISSERTATION
Presented to the Department of Biology
and the Graduate School of the University of Oregon
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
December 2008
11
University of Oregon Graduate School
Confirmation of Approval and Acceptance of Dissertation prepared by:
Marc Dorfman
Title:
"Identifying Roles for Non-Essential Genes in Essential Processes"
This dissertation has been accepted and approved in partial fulfillment of the
requirements for the Doctor of Philosophy degree in the Department of Biology by:
Christopher Doe, Chairperson, Biology
Bruce Bowerman, Advisor, Biology
Judith Eisen, Member, Biology
Karen Guillemin, Member, Biology
Tom Stevens, Outside Member, Chemistry
and Richard Linton, Vice President for Research and Graduate Studies/Dean of the
Graduate School for the University of Oregon.
December 13, 2008
Original approval signatures are on file with the Graduate School and the University of
Oregon Libraries.
© 2008 Marc David Dorfman
111
in the Department of Biology
Marc David Dorfman
An Abstract of the Dissertation of
for the degree of
to be taken
IV
Doctor of Philosophy
December 2008
Title: IDENTIFYING ROLES FOR NON-ESSENTIAL GENES IN ESSENTIAL
PROCESSES
Approved: _
Dr. Bruce Bowerman, Advisor
My dissertation has focused on identifying functions for non-essential genes in
essential process, using the early C. elegans embryo as a model system. The fully
sequenced C. elegans genome contains ~19,800 protein coding genes of which about half
have identifiable homologs in humans. Classical forward genetic mutagenesis screens,
and more recently, genome-wide RNA interference (RNAi) screens has led to the
identification of most essential genes in the genome. Analysis of the phenotypic data
from mutants and RNAi screens shows that roughly 15% of the genes are essential and an
additional 15% produce some other easily identifiable knockdown phenotype. This leaves
about 70% of genes that have no functional information. Genetic modifier screening
allows for the identification of roles for genes that do not produce a loss of function
phenotype on their own but are able to modify the phenotype of a specific mutant. In my
first chapter, I introduce approaches to identifying new gene functions and the usefulness
vof C. elegans as a model system in this pursuit. In Chapter II, I describe a type of high-
throughput genetic modifier screen that combines the sensitized genetic background of
temperature-sensitive (ts) embryonic lethal mutants, and RNAi, to identify genes that
either enhance or suppress embryonic lethality seen in the mutant background. I also
summarize results from screening four ts mutants using this method. The following two
chapters describe the identification and characterization of genetic modifier genes for two
different ts embryonic-lethal mutants. Chapter III describes modifiers of rfl-l, a
conserved gene required for proper cytoskeletal regulation in the early C. elegans
embryo. Chapter IV describes modifiers of lit-I, also a conserved gene, that is required
for regulation ofWnt signaling and cell fate specification in C. elegans. These findings
reveal novel genetic interactions and provide functional information about many
conserved but non-essential genes that have had no previous characterization.
Conclusions are also made about the effectiveness ofts mutant/RNAi screening in the
pursuit of identifying new gene functions.
This dissertation contains co-authored material that has been previously
published, and material that is currently in review, or is being prepared for publication.
CURRICULUM VITAE
NAME OF AUTHOR: Marc David Dorfman
PLACE OF BIRTH: Santa Barbara, California
DATE OF BIRTH: July 17, 1979
GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED:
University of Oregon, Eugene, Oregon
University of California at Santa Barbara, California
DEGREES AWARDED:
Doctor ofPhilosophy in Biology, 2008, University of Oregon
Bachelor of Science, 2002, University of California at Santa Barbara
AREAS OF SPECIAL INTEREST:
Cell Biology
Genetics
Developmental Biology
PROFESSIONAL EXPERIENCE:
Graduate Research Fellow, Department ofBiology, University of Oregon,
Eugene, Oregon, 2004-2008
Technology and Entrepreneurship Program Fellow, Lundquist College of
Business, University of Oregon, 2008
Graduate Teaching Fellow, Department ofBiology, University of Oregon,
Eugene, Oregon, 2002-2003
Researcher, Dr. Joel Rothman Lab, University of California at Santa
Barbara, 2002
VI
Vll
Laboratory Technician, Qualtek Molecular Laboratories, Goleta, California, 1998
-2001
GRANTS, AWARDS AND HONORS:
NIH Developmental Biology Training Grant, University of Oregon, 2004-2007
Co-Host of 5th Annual Developmental Training Grant Symposium
"(Re)Generating Stem Cells", University of Oregon, June 2007
President of Students in Biological Sciences (SIBS), University of Oregon, 2004-
2005
PUBLICATIONS:
Dorfman, M.D., S.M. O'Rourke, J. Gomez and B. Bowerman, (2008) Using RNA
interference to identify modifiers ofa temperature-sensitive, embryonic-lethal
mutation in the essential C. elegans gene rjl-l. Genetics (In Review).
O'Rourke, S. M., M. D. Dorfman, J. C. Carter and B. Bowerman, 2007 Dynein
modifiers in C. elegans: light chains suppress conditional heavy chain mutants.
PLoS Genet 3: e128.
V11l
ACKNOWLEDGMENTS
I wish to thank my advisor, Dr. Bruce Bowerman, for always putting my best
interests first and for providing excellent guidance and encouragement throughout my
graduate school career. I also acknowledge all my lab members past and present for
helping to create a positive work and learning environment. Specifically I would like to
thank Dr. Sean O'Rourke. Sean is a brilliant scientist who was always generous in
providing training, guidance and expert opinion. Working closely with him has greatly
enhanced my scientific training. I would like thank Graduate Program Coordinator Donna
Overall for always being vigilant about important deadlines and reminding me whenever
I needed to complete something. Lastly, I thank Dr. Dan Graham for his help when the
chips were down and a computer or a piece of equipment needed to be fixed in a hurry.
--------- ------
IX
I co-dedicate this document to my parents and to my wife lana, who have supported my
endeavors, scientific and otherwise.
xTABLE OF CONTENTS
Chapter Page
I. INTRODUCTION TO FUl\JCTIONAL GENOMIC ANALYSIS USING THE
MODEL SYSTEM CAENORHABDITIS ELEGANS 1
•II. A GENETIC MODIFIER SCREEN USING HIGH-THROUGHPUT RNAI Al\ID
TEMPERTURE-SENSITIVE, EMBRYONIC-LETHAL MUTANTS.................. 9
Introduction....... 9
Materials and Methods 11
Results 13
Discussion 15
III. USING RNA INTERFERENCE TO IDENTIFY MODIFIERS OF A
TEMPERATURE-SENSITIVE, EMBRYONIC-LETHAL MUTATION IN
THE ESSENTIAL C. ELEGANS GENE RFL-l...................................................... 20
Introduction 20
Materials and Methods 24
Results 27
Discussion 45
IV. NOVEL REQUIREMENTS FOR THE GENES YI19CIA.l AND DRH-3 IN
WNTIMAPK SIGNALING PATHWAYS AND ENDODERM
SPECIFICATION IN C. ELEGANS............................................................. ............ 53
Introduction 53
Materials and Methods 57
Results 58
Discussion 67
V. CONCLUSIONS 73
APPENDIX: SUMMARY OF RFL-l(OR198TS) MODIFIERS...................................... 77
REFERENCES 79
Xl
LIST OF FIGURES
Figure Page
Chapter II
1. Temperature versus embryonic viability for spn-4(or191) and zen-4(or153) ... 14
Chapter III
1. Temperature versus embryonic viability for rjl-1 (or198) and wildtype (N2) ... 29
2. Enhancement ofrjl-1(or198ts) embryonic lethality.......................................... 31
3. Specificity of modifiers for rjl-1(or198ts) embryonic lethality......................... 33
4. Suppression of early embryonic cell division defects in rjl-1 (or198ts)
mutants.............................................................................................................. 36
5. Expression of GFP fusion to suppressor encoded proteins in early embryos
from transgenic lines.. 40
6. Spinning-disk confocal projected Z-stack images ofGFP and tdTomato
fusion proteins.................................................................................................... 42
7. Nuclear localization of GFP::RFL-1 is lost in ula-1 (RNA i) but not in other
neddylation pathway defective backgrounds. 45
Chapter IV
1. Suppression of lit-1 (or131ts) lethality by RNAi 61
2. Specificity analysis of genetic interactions caused by Yl19C1A.1 or drh-3
RNAi 63
3. RNAi knockdown ofYl19CIA.1 (A) and drh-3 (B) effect on embryonic
viability in three different ts alleles of lit-1 and one ts allele ofmom-4 64
4. Gut granules serve as a marker for endodermal development........................... 66
5. Expression ofGFP fusion to Yl19C1A.1 encoded protein in early embryos
from transgenic strain. 68
xu
LIST OF TABLES
Figure Page
Chapter II
1. Screening output................................................................................................ 14
Chapter III
1. Modifier depletion does not enhance early embryonic cell division defects
in rfl-l (or198ts) mutants................................................................................... 38
Chapter IV
1. Percent embryos with gut granules 66
1CHAPTER I
INTRODUCTION TO FUNCTIONAL GENOMIC ANALYSIS USING THE
MODEL SYSTEM CAENORHABDITIS ELEGANS
In recent years many whole genome sequencing projects have been completed,
including that of humans, providing us with the DNA sequences that encode all of the
heritable factors necessary for life. While the completion of these sequencing projects are
heroic and important achievements, we are far from understanding the functions and
requirements of most of the genes in anyone of those genomes. Model organisms that are
amenable to experimental genetic analysis are essential for identifying the functions of
specific genes. With the availability of whole genome sequences, these functions can be
extrapolated to understanding the roles of genes with similar sequences in humans.
One interesting finding from sequencing data and subsequent analysis, was the
relatively small number of predicted gene coding sequences in humans and other multi-
cellular organisms compared with previously sequenced genomes of single celled
organisms. The relatively small number (25,000 for humans vs 6000 for the single celled
yeast), did not seem to account for the vast complexity of regulatory processes required
for the development and maintenance of multi-cellular organisms and complex
phenotypes (LANDER et al. 2001). Alternative-RNA splicing (SCHELLENBERG et al.
2008), post-translational modification (MEINNEL and GIGLIONE 2008), and epigenetics
(GUlL and ESTELLER 2008), have all been shown to play important roles in exploiting the
2limited repertoire of heritable factors to allow for the development of higher organisms.
Moreover, the identification of genetic interaction networks, how genes affect the
regulation and function of other genes, will no doubt be essential in understanding
fundamental biological processes and the development of complex traits. In addition,
genetic interactions can provide important insight into the function of individual genes.
Importantly, genetic interactions can reveal roles for genes that do not have an easily
detectable loss-of-function phenotype of their own and therefore have avoided detection
in previous genetic mutagenesis studies. The model system C. elegans has emerged as
one of the most important model organisms in the pursuit of identifying such genetic
interactions.
Genetic analysis in the model organism Caenorhabdidtis elegans: Caenorhabditis
elegans is an ideal model organism for genetics study as it is complex enough to study
development, behavior and specific disease models, but simple to maintain and amenable
to various methods of forward and reversed genetics to identify gene function. The
genome sequence of C. elegans released in 1998 (c. elegans Sequencing Consortium),
was the first to be released of any multicellular organism. Analysis and comparison with
the human genome sequence has predicted that roughly 50% of human genes have
identifiable homologs in C. elegans (Wormbook 2005). Another import characteristic of
C. elegans is that embryos and adults are transparent and therefore their invariant
development to produce the adult animal consisting of 959 somatic cells, can be easily
monitored with a light microscope. This allows for the identification of aberrant
3phenotypes at a cellular level which can provide information about the specific function
of genes when they are reduced in function.
. It has been over three decades since Sydney Brenner published the first genetic
screen in C. elegans. This paper describes the use of a chemical mutagen to produce
random mutations throughout the genome (BRENNER 1974). Then, by screening through
the F2 generation he identified 619 mutants with visible phenotypes which were then
grouped and classified into distinct phenotype classes. This screen is an example of a
"simple screen" where the experimenter's goal is to find mutations that cause any type of
abnormal phenotype. This screen was critical for establishing phenotypic classes such as
uncoordinated (unc), small body (sma), dumpy (dpy), long body (Ion) and rolling
locomotion (rol). Researchers later used these phenotype classes to identitY mutations in
different genes that caused the same or similar phenotype and thereby began to establish
genetic pathways.
In the years since Syndey Brenners's landmark paper, researchers have designed
and executed many different types of forward genetic screens, and this has led to the
characterization of many thousands of genetic mutation alleles covering roughly 5,000
individual genes. Many screens have been designed to identitY specific phenotypes such
as maternal embryonic lethal phenotypes (mel). Maternal embryonic lethal mutants
describe mutations in genes that are essential for embryonic development, but embryos
homozygous for the mutation, but that have wildtype, maternally derived gene product
(from heterozygous parent), are viable.
4Selectable markers can be used to aid in the identification and/or the isolation of specific
phenotypes such as mel. For example, mel screens have been done using an mutant allele
of the gene lin-2 that causes an egg laying defect (PRIESS et al. 1987). Embryos that have
maternally deposited wildtype mel gene product are viable and will hatch inside of the
adult hermaphrodite lin-2 mutants, leading to the distinctive "bag of worms" phenotype,
instead of being passed through the vulva. Embryos from homozygous mel mutant worms
will fail to hatch but will remain inside the mother. While mutants obviously cannot be
recovered from the worm containing dead eggs, heterozygotes containing the mel
mutation can be isolated from siblings of the adult contained on the same plate. Thus
selectable markers aid in reducing the amount of labor and time required to conduct
large-scale screens.
Functional genomics using genome-wide RNA interference: Reducing gene function
to identify phenotypes and therefore gene functions in C. elegans has benefitted greatly
from the advent of RNA interference, the ability of double stranded RNA (dsRNA) to
target and degrade endogenous mRNA, thereby thwarting protein production. First
applied by microinjection (Guo and KEMPHUES 1995), RNAi can now be performed by
feeding worms E. coli that expresses dsRNA corresponding to a particular gene
(TIMMONS and FIRE 1998). This method has allowed for genome-wide RNAi screens to
be performed routinely using various genetic backgrounds (KAMATH and AHRINGER
2003; MAEDA et al. 2001; SIMMER et al. 2003; SONNICHSEN et al. 2005). Data derived
from these genome-wide RNAi screens and decades of forward mutagenesis screens have
5led to the discovery of most if not all essential genes in the genome. Current estimates put
the number of essential genes at roughly 15% ofthe genome while another 15% produce
some other readily identifiable phenotype when depleted with RNAi. The remaining 70%
of genes (about 14,000) have no known loss-of-function phenotype. Studies using
deletion mutants in yeast Saccharomyces cerevisiae show a similar ratio of essential
(19%) to non-essential genes. About one third of these 14,000 C. elegans genes have
counterparts in mammalian organisms, and therefore it will be a worthwhile pursuit to
elucidate functions for many of these genes. The challenge is finding the best way to
identify such functions in an efficient manner. Various methods of genetic modifier
screening will likely playa major role in such efforts.
The role of genetic modifier screening in determining gene functions: Genetic
modifier screening is a powerful method to identify genes that function in or influence
specific molecular pathways. By identifying second site mutations that either enhance
(worsen) or suppress (alleviate) a particular phenotype, researchers can accumulate
mutants, quantify their effect on the background phenotype, and then clone and
characterize the mutated loci. Often, modifier screens can reveal functions for genes that
do not have a loss of function phenotype of their own, because of genetic redundancy,
genetic buffering or because the phenotype is too subtle to detect. But, when combined
with a background mutation, certain mutants of non-essential genes display a synthetic
phenotype. This is sometimes referred to as "sensitized screening" as it allows for
detection of mutant phenotypes that would not be penetrant enough, or completely silent,
6in an otherwise wildtype background. Reverse genetic screens using RNAi offers an
attractive alternative to identifying second site modifiers, as it allows the researcher to
perform targeted screens of suspected genetic modifiers, or large-scale, high-throughput
screens to identify many candidate modifier genes, without the need for genetic mapping
and sequencing to identify modifier loci as with forward genetics screens.
Temperature-sensitive mutations offer a unique and ideal background for genetic
modifier screens as mutants of essential genes can be maintained at permissive
temperature, and then raised at semi-permissive temperatures for the detection of
enhancers and suppressors. The ability to control the function of ts mutants proteins by
altering temperature also allows the researcher to identify requirements of the protein at
different times during development. For example, a loss-of-function mutant of a gene that
is essential for very early embryonic development likely would lead to a tenninal
phenotype before most of the embryonic cell divisions and morphogenesis has occurred.
This would prevent the detection of functional requirements for the gene in later stages of
development. With ts mutations, one can circumvent early requirements by maintaining
the animals a pennissive temperature and then "upshifting" to a non-permissive
temperature later in development.
High-throughput genome-wide modifier screens necessitate a method of
qualitative scoring that is relatively quick and accurate. Temperature-sensitive mutants
are especially useful for high-throughput, qualitative screening as they can be raised at
temperatures that both maximize genetic sensitivity and the ability to detect changes in
embryonic lethality. For example, by growing a ts embryonic-lethal mutant at a
7temperature at which only 2% of progeny hatch with control RNAi, makes a suppressor
that increases viability by say 8%, a five fold increase in viability and therefore will
likely be detected. Detection of slight changes in lethality may not be detectable at more
intermediate temperatures. Furthermore, most ts mutants display higher degree of
phenotype variability at intermediate temperatures compared with temperature near the
points of 0% orlOO% penetrance.
In the following chapters, a novel type of high-throughput RNAi modifier screen
is introduced and results of screening are presented. Chapter II contains published, co-
authored material describing the screening procedure. Chapters III and IV present
coauthored, in-depth analysis and characterization of genetic loci that modify the ts
mutants rfl-1 (or189ts) and lit-1 (or131ts). The gene rfl-1 encodes a conserved gene
required for proper cytoskeletal regulation in the early C. elegans embryo. The gene lit-1
encodes a conserved gene that is required for regulation of Wnt signaling and cell fate
specification in C. elegans. Together, a total of 59 reproducible genetic modifiers were
identified for these two mutants. The majority of these genes have no previously
identified function while about half have homologous counterparts in humans. The
identification of novel roles for these genes and the preliminary work in characterizing
some of the conserved but non-essential genes, will hopefully aid in the pursuit of
understanding how essential biological pathways that control cell division and cell fate-
patterning work. Moreover, non-essential genes that influence essential processes may
prove to be valuable drug targets as altering their function may allow for modification of
an essential process without causing deleterious side effects.
8Bridge to Chapter II: In the preceding chapter, I introduced various approaches to
identifying gene function, the usefulness of C. elegans in the study of functional
genomics, and the attributes of temperature-sensitive mutants in genetic studies. In
chapter II, I will present a novel screening approach using feeding RNAi and ts mutants
to identify roles for non-essential genes in essential processes. I also present some results
of screening four ts mutants, and discuss the overall effectiveness of the screening
approach. The chapter contains some material from a previously published, coauthored
publication.
9CHAPTER II
A GENETIC MODIFIER SCREEN USING HIGH-THROUGHPUT RNAi AND
TEMPERTURE-SENSITIVE, EMBRYONIC-LETHAL MUTANTS
The high-throughput screening procedure ofts mutants with feeding RNAi
described in this chapter, was developed primarily be three lab members: Jose Gomez,
Sean O'Rourke and myself. Jose Gomez conceived ofthe idea to use ts mutants to screen
for both enhancers and suppressors of embryonic lethality. Sean O'Rourke played a
major role in the development of the high-throughput procedure. Along with Sean
O'Rourke I contributed to optimizing the screening procedure and also performed all
screening and subsequent analysis of the mutants presented in this chapter.
INTRODUCTION
Functional genomic analysis of Caenorhabditis eiegans has been accelerated in
recent years by the ability to inhibit the expression of targeted genes using RNA
interference (RNAi), the delivery of dsRNA into cells which causes a degradation of
endogenous mRNA corresponding to the sequence of the dsRNA. In C. eiegans RNAi
can be performed by three different methods: soaking (MAEDA et al. 2001), injection
(SONNICHSEN et al. 2005),or feeding (TIMMONS et ai. 2001). With the availability of
----------------------
10
whole genome sequence data released in 1998, researchers have already conducted
genome-wide RNAi screens using each of the three methods ofdsRNA (KAMATH and
AHRINGER 2003; MAEDA et al. 2001; SIMMER et al. 2003; SONNICHSEN et al. 2005). In
addition, genome-wide RNAi screens have been done in mutant backgrounds that have
been shown to be hypersensitive to RNAi, presumable allowing for a more complete
knockdown of genes that are relatively insensitive to the effects ofRNAi (SIMMER et aI.
2003). The data derived from these screens have lead to the identification of most if not
all essential genes and genes that cause an easily identifiable visible phenotype (see
Chapter 1 for more detailed discussion). About 15% of the 20,000 genes in the genome
produce an embryonic lethal phenotype while another 15% produce some other readily
identifiable abnormality. The remaining 70% of genes (roughly 14,000) have no known
function, even though roughly one third of these genes have identifiable homologues in
mammalian genomes. Identifying roles for these genes presents a challenge to
researchers, but it seems to be a challenge worth pursuing as it is likely that many of the
genes in this "silent majority" have important roles in major biological processes even if
they lack a clear loss-of-function phenotype of their own.
Modifier screens using sensitized genetic backgrounds present the opportunity to
identify roles for non-essential genes in essential processes. In this study, we have
developed a novel method of high-throughput modifier screening using feeding RNAi
with animals carrying temperature-sensitive (ts), embryonic-lethal mutations to identify
genes that modify the degree of embryonic lethality normally present in the mutant
background. The use of ts alleles allowed for the identification of both genetic enhancers
11
and suppressors by altering the incubation temperature to a semi-permissive (for
identifying enhancers) or just non-permissive temperature (for identifying suppressers)
during the screening process. We describe the systematic screening process which
includes three screening steps (primary, secondary and tertiary), and present results of
screening four different ts mutants; spn-4 (ori9its); zen-4(ori53ts); lit-i (ori3lts) and
rfl-i (or i98ts), each defective in distinct processes essential for early embryonic
development. We go on to discuss some of the candidate modifiers identified for spn-
4(ori9its) in the discussion section; modifiers of rfl-i (or198ts) and lit-i (or13its) are
discussed and analyzed in chapters III and IV, respectively.
MATERIALS AND METHODS
Screening tools and basic procedure: To identify genes that, when reduced in function,
can suppress or enhance conditional ts mutants, we developed a high-throughput RNAi
based screen. To reduce gene function we used a library of 16,757 bacterial strains that
each express dsRNA corresponding to an exon-rich gene sequence. After obtaining the E.
coli RNAi library from the MRC Geneservice (Cambridge, UK), we rearrayed it into a
48-well microplate format using a liquid-handling Qiagen BioRobot 8000. E. coli strains
were thawed from -80 DC storage and inoculated into 1 ml of LB + 100 mg/ml
ampicillin-containing 96-well growth plates (Whatman) and covered with microporous
sealing film (USA Scientific). Only 48 wells of the 96-well growth plates were filled with
12
media, corresponding to the rearrayed E. coli library. After overnight shaking incubation
at 37°C, 20 /-ll of the cultures were dispensed with a 24-channel electronic repeating
pipette (Rainin) onto 48-well plates (Nunc) containing NGM agar, 100 /-lg/ml ampicillin,
and 1 mM IPTG and allowed to dry and induce dsRNA at 37°C overnight. The 48-well
agar plates were filled using a Wheaton Unispense peristaltic pump equipped with a
custom-made adaptor (University of Oregon Technical Science Administration) that
allowed simultaneous filling of eight wells with the agar solution. Approximately 15
hypochlorite-synchronized Ll mutant larvae were pipetted into each well of the 48-well
plates with a multichannel pipette and allowed to produce broods. Screening for Fl
viability was performed by visual examination with a stereomicroscope. Phenotypes were
recorded on a Microsoft Excel spreadsheet.
Identifying candidate modifiers and testing for reproducibility: primary, secondary
and tertiary Screens: During the primary screening process, a non-stringent scoring
criterion for identifying putative enhancers and suppressors was used to reduce the
possibility of failing to detect potentially interesting genetic interactions. To eliminate
false-positive interactions, and to identify the most reproducible and penetrant genetic
interactions worthy of further study, each positive interaction was systematically re-
screened in both a "secondary" and "tertiary" screening process (Table 1). During the
secondary screening process each interaction was retested using the same method as in
the primary screening process. Only the genetic interactions that exhibited a reproducible
effect were carried on to tertiary screening. During the tertiary screen each RNAi clone
13
that exhibited a reproducible genetic interaction was streaked out, and two colonies were
selected for retesting. This procedure eliminated the possibility that two different RNAi
clones, accidentally mixed in the library, were acting together to interact with the
background mutant. Results from the tertiary screening process are also included in Table
1.
RESULTS
A sensitized genetic background: To identify semi-permissive temperatures that
optimized our ability to identify enhancers and suppressors, we quantified embryonic
viability for embryos derived from spn-4(or191 ts) and zen-4(or153ts) animals grown at 5
different temperatures between 15°C and 25°C (see chapters III and IV for rfl-1 (or198ts)
and lit-1(or131ts) ts curves) (Figure 1). We chose to screenspn-4(or191ts) at 17°C for
enhancement and 23°C for suppression. For the mutant zen-4(or153ts) we chose 15°C for
enhancement and 18°C for suppression.
Candidate modifiers from qualitative screening: We screened four ts mutants using
two separate conditions against a feeding RNAi library to identify enhancers and
suppressors of embryonic lethality. The primary screen yielded 2101 total genetic
modifiers which were consolidated and rescreened for reproducibility using the same
procedure. This secondary screen reduced the number of candidate modifiers to 585. A
final round of screening using bacterial cultures started from single colonies yielded 189
14
spn-4(or191)
30
20
10
O'-------'----...I....---....L..----'------'-----:----'
100
.0 90
.....
~ 80
-5
~ 70
u
'S 60
o£ 50
S
~ 40
~
Figure 1.-Temperature versus embryonic viability for spn-4(or191) and zen 4{or153).
L1 larvae were raised to adulthood at the indicated temperatures on L4440 empty-vector
control bacteria. Progeny were scored as hatched larvae or dead embryos (n>200
embryos for each temperature).
TABLE 1
Screening output
mutant lit-1 Iit-1 spn-4 spn-4 rfl-1 rll-1 ~n-4 zen-4
enh supp enh supp enh supp enh supp
% library 77 94 S4 r79 49 84 ~6 68
~creened/ # of 12874 15716 9029 13208 8192 14045 ~363 11370
genes
Modifier 328 239 ~71 180 248 388 ~20 27
~andidates
primary screen
Reproducible 95 31 135 ~O 101 104 ~5 4
modifiers
~econdary screen
Tertiary screen 89 ~ 21 16 ~2 ~6 Not 0
positives completed
15
modifiers [zen-4(or153ts) enhancer tertiary screening not completed]. Among these
reproducible candidate modifiers are 142 enhancer genes which had no previously
identified essential requirements in genome-wide RNAi screens. Thus, our screen was
able to identify potential novel genetic interactions and functions for non-essential genes.
RNAi knock-down of the gene oma-2 dramatically suppresses embryonic lethality in
spn-4(or191ts) mutants: Enhancer and suppressor screening of spn-4(or19lts) yielded
21 candidate enhancers and 16 candidate suppressors. Among the enhancers are three
genes that are highly conserved and have no RNAi or mutant phenotype in any of several
previously published screens [in our screening process we only eliminated enhancer
candidates that showed lethality in either a genome wide-feeding RNAi screen (KAMATH
and AHRINGER 2003), or genome-wide soaking RNAi screen (MAEDA et al. 2001)].
These genes are H17B01.4, F12AlO.7 and C45G9.1. H17B01.4 and F12AlO.7 both
encode uncharacterized proteins whereas C45G9.1 encodes a predicted casein kinase. Of
the 16 suppressor candidates, one gene stood out during qualitative screening as a
particularly penetrant suppressor. Sequence verification of the RNAi clone confirmed
that it was the gene oma-2. Quantitative analysis of embryonic viability in spn-4(or19lts)
mutants showed a dramatic amount of suppression, from 6% viability with negative
controls, to 59% viability of animals fed oma-2 RNAi. At an even more restrictive
temperature of 24°C, at which only 0.5% of spn-4(or19lts) embryos hatch, viability was
increased to 41 % with oma-2 RNAi. Importantly, oma-2 was not detected as a suppressor
in any of the other three mutants screened in this report, supporting the possibility that
oma-2 has a direct role in spn-4 related processes.
DISCUSSION
16
The goal of the work described here was to conduct a high-throughput RNAi
modifier screen using multiple genetic backgrounds in parallel to detect reproducible
enhancers and suppressors. Using the methods and tools presented in this chapter it is
possible to conduct multiple genome-wide screens in parallel in as little as five weeks.
The methods used have also proven to be a useful approach in identifying roles for non-
essential genes in essential processes.
Screening procedure analysis: The work presented in this chapter took roughly two
years to complete. Much of that time could have been eliminated had we not faced
setbacks due to equipment problems, inactivation ofIPTG, and recurrent bacterial and
fungal contamination. Although equipment failure is not always preventable, extreme
care should be taken to prevent contamination of plates and worm stocks during the
procedure. Solutions containing synchronized L1 larvae should always be tested for
contamination by aliquoting a small volume onto an NGM + 4xAMP plate and
incubating overnight at 37°e. If any colony growth is observed, the solution should not
be used, as even a very small amount of contamination will likely grow rapidly on the
RNAi plates and negate results. All solutions containing IPTG should be immediately
17
frozen in single use aloquots. There is lack of documentation regarding IPTG shelf life
and we found that activity of IPTG is very sensitive to freeze-thaw cycles or storage at
temperatures above freezing. Furthermore, positive and negative controls should be
performed during every RNAi assay to make sure the feeding RNAi is working. Lastly,
it is important to allow bacterial cultures seeded onto agar to completely dry, as a liquid
environment for the worms significantly alters hatching rates. Likewise, care should also
be taken to prevent excessive drying of the agar as this can also affect hatching rates.
Candidate modifiers of spn-4(or191ts): The gene spn-4 encodes a protein containing an
RJ'JP-type RNA-binding domain and is required for several processes in the early C.
elegans embryo including proper cell fate patterning and mitotic spindle orientation
(GOMES et al. 2001; LABBE and GOLDSTEIN 2002). Loss of function of spn-4 ultimately
leads to reduced gut and pharyngeal tissue and excess body wall muscle and germline
(GOMES et al. 2001). We identified one highly penetrant suppressor of spn-4(or191ts)
mutants, that did not suppress lethality in any of the other three mutants in this study
(qualitative analysis). RNAi knock-down of oma-2 increased viability in spn-4(or191ts)
mutants from 6% to 59% viability.
The gene oma-2 is at least partially redundant with its paralog oma-1 and has 63%
amino acid similarity. Neither gene is essential, but double mutants have severe defects in
oocyte maturation often leading to sterility (DETWILER et al. 2001). oma gene products
encode CCCH-type zinc finger proteins that, like SPN-4, have been shown to bind to
maternal germline mRJ'JA targets and to regulate their expression. A recent study showed
18
that SPN-4 and OMA-l/2 have at least one mRNA target in common as both bind the
3'UTR of the maternal mRNA nos-2 and suppress its translation (JADHAV et al. 2008).
Requirement for spn-4 has been identified in several studies examining the establishment
of polarity in the early C. elegans embryo (GOMES et al. 2001; JADHAV et al. 2008;
LABBE and GOLDSTEIN 2002), and it has become clear that both spn-4 and oma-l/2 have
multiple different roles that influence these early events in development. The data
presented here suggest that oma-2 and spn-4 may have some opposing role(s) which
would explain why depleting oma-2 would suppress lethality in a spn-4 loss-of-function
mutant. It is not known ifRNAi to oma-2 is specific, or if oma-l gene product is also
partially depleted because of sequence homology. It would be interesting to cross oma-l
and oma-2 mutant alleles into the spn-4(or19lts) background and test whether lethality is
enhanced or suppressed. A significant phenotype difference between these double
mutants could indicate separate functions for oma-l and oma-2. Additionally, it would be
interesting to identify all or most of the mRNA targets of these proteins. This information
will likely explain the mechanism of the interaction identified in this RNAi screen.
Bridge to Chapter III: I identified many reproducible interactions using the screening
approach outlined in the proceeding chapter. In chapter III, I present coauthored material
currently in review (Genetics) describing a systematic analysis of enhancers and
suppressors of the gene rfl-l. The analysis includes quantification of enhancement or
suppression, a thorough specificity analysis of each modifier, protein localization data of
several conserved and specific modifiers, and analysis of cellular phenotypes in enhanced
19
or suppressed rfl-l mutant embryos. Results of this work indicate that many of the
genetic modifiers identified likely play novel and important roles in early embryonic cell
division. Important conclusions are also made about assessing specificity of genetic
interactions before formulating hypothesis about potential protein functions.
20
CHAPTER III
USING RNA INTERFERENCE TO IDENTIFY MODIFIERS OF A
TEMPERATURE-SENSITIVE, EMBRYONIC-LETHAL MUTATION IN THE
ESSENTIAL C. ELEGANS GENE RFL-l
Reproduced with permission from: Marc Dorfman, Jose-Eduardo Gomes*,
Sean O'Rourke* and Bruce Bowerman
*These authors contributed equally to this manuscript
Manuscript currently in review at Genetics
INTRODUCTION
Ubiquitin-mediated proteolysis regulates many biological processes O'JANDI et ai.
2006). These include, in the nematode Caenorhabditis eiegans, the early developmental
processes of oocyte maturation, cell cycle progression, cell polarization, and cell fate
patterning, all of which require the timely destruction of maternally expressed proteins
(BOWERMAN and KURZ 2006). One C. eiegans protein targeted for proteolysis early in C.
eiegans embryogenesis is MEl-I, the AAA-ATPase subunit of the microtubule severing
complex called katanin (Dow and MAINS 1998; KURZ et ai. 2002; MAINS et ai. 1990;
PINTARD et ai. 2003a; SRAYKO et ai. 2000; Xu et al. 2003). Katanin is a heterodimer of
two subunits called p60 and p80 in vertebrates and MEI-l and MEI-2 in C. eiegans.
Katanin in C. eiegans is required for proper assembly and function of the small, barrel-
21
shaped meiotic spindles (ALBERTSON and THOMSON 1993; McNALLY et ai. 2006), but it
must be degraded after meiotic divisions to permit assembly ofthe much larger first
mitotic spindle in the one-cell zygote. In mutants that fail to degrade katanin after the
completion of meiosis, the first mitotic spindle is fragmented and mis-oriented,
cytokinesis is defective, and the embryos die without hatching (Dow and MAINS 1998;
KURZ et ai. 2002; SRAYKO et al. 2000).
The katanin subunit MEI-1 is targeted for poly-ubiquitylation and proteolytic
destruction by a Cullin-based E3 ligase. This complex includes the Cullin scaffolding
protein CUL-3 and a substrate specific adaptor called MEL-26 that binds to CUL-3
through a BTB domain and to MEI-1 through a MATH domain (PINTARD et ai. 2003b).
Cullin 3-based E3 ligases in mammals also utilize substrate-specific adaptor proteins that,
like MEL-26, have both a Cullin-binding BTB/POZ domain and another protein/protein
interaction domain that binds to the substrate (ANGERS et al. 2006; CULLINAN et ai. 2004;
GEYER et ai. 2003). While MEI-1/Katanin down regulation by the CUL-3/MEL-26 E3
ligase is essential at most growth temperatures, a mei-26 null mutation is viable at the low
growth temperature of 15°C (Lu and MAINS 2007). This bypass ofmei-26 at 15°C
depends at least in part on the anaphase promoting complex and its targeting ofMEI-1
for proteolytic degradation (Lu and MAINS 2007). Phosphorylation by the kinase MBK-2
primes MEI-1 for proteolysis (QUINTIN et al. 2003; STITZEL et ai. 2007), and also
promotes the down-regulation of MEI-1 by the anaphase promoting complex (Lu and
MAINS 2007).
22
CUL-3 is the only C. eiegans Cullin thus far identified that requires modification
by the ubiquitin-like protein Nedd8 (BOWERMAN and KURZ 2006) In contrast, C. eiegans
CUL-2 is required for progression through meiosis, and for the localized degradation of
cell fate determinants in one-cell stage embryos, but neddylation-defective mutants do
not exhibit these early defects (BOWERMAN and KURZ 2006). The neddylation of CUL-3
is mediated by a Nedd8 protein conjugation pathway. It begins with a heterodimeric E1
activating enzyme consisting ofULA-1 and RFL-1 (Uba3p in budding yeast), and also
includes the E2 conjugating enzyme UBC-12 (JONES and CANDIDO 2000; KURZ et ai.
2002; SRAYKO et ai. 2000) and the E3 ligase DCN-1 (KURZ et ai. 2005).
The down-regulation of MEl-l/katanin by the CUL-3/MEL-26 E3 ligase requires
a balance of both CUL-3 neddylation, mediated by the Nedd8 conjugation pathway, and
de-neddylation, mediated by the highly conserved COP-9 Signalosome (PINTARD et al.
2003a). Other Cullin based E3 ubiquitin ligases also require a balance ofneddylation and
de-neddylation (BORNSTEIN et ai. 2006; HETFELD et al. 2008; LYAPINA et al. 2001;
SCHWECHHEIMER et ai. 2001). De-neddylation may modulate activation of the E3 ligase
and thereby prevent the premature degradation of substrate adaptor proteins that also can
become poly-ubiquitylated and degraded as a result of E3 ligase function.
To identify additional factors that influence neddylation and the down regulation
ofMEI-l/katanin after the completion of meiosis in C. eiegans, we report here our use of
RNA interference (RNAi) to reduce gene functions in a temperature-sensitive
neddylation-defective mutant, rjl-l(or198ts). The discovery ofRNAi and its systemic
properties in C. eiegans have made it possible to systematically target C. eiegans genes
23
for depletion by feeding worms bacterial strains that express double-strand RNAs
corresponding to C. elegans gene sequences (BAUGH et al. 2005; FEINBERG and HUNTER
2003; FIRE et al. 1998; LEHNER et al. 2006; TIMMONS et al. 2001; VAN HAAFTEN et al.
2006). Furthermore, chemical mutagenesis screens have identified temperature-sensitive
mutations in many essential C. elegans genes, providing opportunities to sensitize genetic
backgrounds for synthetic screens by choosing intermediate growth temperatures that
sensitize the genetic background and also optimize quantitative scoring of embryonic
viability. Recently, genome-wide RNAi screens have been used to identify C. elegans
genes that, when reduced in function, restore viability to temperature-sensitive
embryonic-lethal mutants (LABBE et al. 2006; O'ROURKE et al. 2007). As a loss of
suppressor function restores mutant viability, the suppressors may negatively regulate
either the wild-type gene product or the process that requires the wild-type gene product.
Here we report our identification of21 C. elegans genes that, when reduced in
function by feeding RNAi, reproducibly suppressed rjl-1 (or198ts) embryonic lethality
and 32 genes that enhanced rjl-1 (or198ts) lethality. Most ofthe suppressors were specific
for rjl-1 (or198ts): reducing their function did not suppress embryonic lethality associated
with temperature sensitive mutations in four other essential genes. In contrast, specific
enhancement was less common. Finally, many of the rjl-1-specific suppressors and
enhancers are conserved but appear to be non-essential.
24
MATERIALS AND METHODS
C. elegans strains and culture: Strains were cultured according to standard procedures
(BRENNER 1974). Temperature-sensitive mutants were maintained at 15°C, GFP-
expressing strains were maintained at room temperature. Isolation of transgenic worms
was performed with the microparticle bombardment method as previously described
(O'ROURKE et al. 2007; PRAITIS 2006).
RNAi screening and quantification of embryonic viability: Methods used for RNAi
screening and quantification of embryonic viability in this study as those described in
detail in O'ROURKE et al. 2007, with the following modifications. For scoring
enhancement, genes that previously had been identified as lethal when targeted by RNAi,
as reported in large-scale screens, were not given candidate enhancer status (KAMATH
and AHRINGER 2003; SIMMER et ai. 2003) To quantify embryonic viability of embryos at
23.5°C, the broods of7-10 gravid adult worms were analyzed. For all quantitative
analysis, experiments were repeated at least four times and the average viability
determined. For enhancement, we report the percentage of dead embryos (we calculated
this percentage for each replicate and then took the average of the replicates). For
suppression, we report the percentage of viable larvae, calculated again by taking the
average of the replicates. The total number of progeny counted, percent viability or
lethality and standard deviations used to generate Figure 3 and data for wildtype (N2
strain) embryonic viability are included in Supplemental Table 2.
25
Molecular biology: For allpie-1 driven, N-terminal GFP constructs (specifically, the rjl-
1 suppressors), genes were amplified using Pfu Turbo polymerase (Stratagene) from a
cDNA library (Invitrogen), with the exception ofRlOD12.14, which was amplified from
N2 genomic DNA. PCR products were subsequently ligated into pGEM-T or pGEM-T-
easy shuttle vectors (Promega). Inserted genes were sequenced in the University of
Oregon sequencing facility prior to cleavage and ligation into pS026 (pS026 described
in O'ROURKE et al. 2007).
To construct N-terminal GFP and tdTomato (SHANER et al. 2004) fusions for sel-
10, C24DIO.l and rjl-1, standard recombineering methods were used as described in
protocol #3, available at (http://recombineering.ncifcrf.govlProtocol.asp) as previously
described (WARMING et al. 2005). We used the following fosmid clones available from
GeneService Limited (http://www.geneservice.co.uk): WRM061 OaC12 (sel-10)
WRM0633dC (C24DIO.l) and WRM066dF09 (rjl-l). We used pS026 as a template for
amplifying GFP (O'ROURKE et al. 2007). For the gap repair step we used pPUB (SAROV
et al. 2006), and designed primers to allow for inclusion of DNA sequence up to the next
open reading frame (start or stop codon) of the gene of interest. To construct the N-
terminal tdTomato::RFL-l fusion we used the pAA64 Vector to amplify the fluorescent
protein-encoding gene. The detailed recombineering protocol for generating C. elegans
fusion proteins will be described in a subsequent publication; details are available upon
request.
26
GFP imaging: Imaging ofGFP and tdTomato fusion protein localization was done by
mounting embryos or whole worms on M9 + 3% agarose pads on microscope slides and
overlayed with a coverslip. Time-lapse videos were obtained on a spinning disk Leica
DMI 4000B microscope using a Leica 63X/IAO-0.60 HCX Plan Apo oil objective, fitted
with a Hamamatsu EM-CCD Digital Camera. Images in FIGURE 6 are projected stacks
that include optical sections through the entire embryo or worm at spacing ofO.5f!M for
embryos and I f!M for worms. Images of GFP::C24D10.1 in FIGURE 6 D-F were taken
with a Leica 40X/1.25-0.75 Plan Apo oil objective and all other images were taken with
the Leica 63X objective described above. Data was recorded using Velocity software and
videos and images were adjusted for contrast in ImageJ and adjusted for levels with
Adobe Photoshop. ImageJ was used to obtain pixel intensities ofGFP::RFL-1 in nuclei of
unenhanced images (Supplemental FIGURE 1). Measurements were taken by selecting a
circular area slightly smaller than the nuclei and obtaining average intensity values for
each nucleus.
DIe imaging: Cellular phenotypic analysis for suppression was performed by incubating
rjl-l (or198ts) L1 stage larvae at 20°C, followed by a shift to 23°C once the larvae reached
the L4 stage (~60hrs at 20°C). The incubation ofrjl-l (or198ts) mutants at 20°C through
L4 stage allowed for slightly increased brood size and a decrease in the number of worms
displaying a sterile phenotype. To image the embryos at a specific temperature we
utilized a temperature controlled microscope stage, equipped with an HEC-400 Heat
Exchanger (20/20 Technology) and a BC-11O Bionomic Controller (20/20 Technology).
27
To calibrate the apparatus we used an Omega HH12 temperature probe to acquire the
temperature of a M9 + 3% agarose pad placed on a sapphire inset metallic microscope
slide (20120 Technology), while on the microscope stage with the light source turned on.
We adjusted the Bionomic controller until a temperature of23°C was achieved. Use of
the temperature-controlled stage prevented us from achieving optimal focus of the
condenser and therefore we were unable to obtain high contrast DIC images, although
image quality was sufficient for analysis of phenotypes.
Following incubation at 23°C for 12 hrs embryos were mounted on the sapphire
crystal inset/metal microscope slides and immediately transferred to the microscope stage
Images were recorded every 5 s using aDage MT1 VE1000 digital camera and Scion
Image or ImageJ software. Contrast and levels were adjusted using Adobe Photoshop.
Spindle angle measurements were done using the ImageJ angle tool. Spindle angles at
cytokinesis were measured one minute (12 frames) following first appearance of a
cytokinetic furrow. The same procedure was used to image embryos for enhancement of
cellular phenotypes except incubation of mutant animals was done at a constant
temperature throughout development as brood size was not significantly reduced at these
less restrictive temperatures (17°C, 18°C or 20°C). The microscope stage temperature was
calibrated to these lower temperatures for imaging as described above.
RESULTS
A sensitized genetic background: To identify semi-permissive temperatures that
optimized our ability to identify enhancers and suppressors of rfl-l (or198ts) embryonic
28
lethality, we examined the viability of embryos produced by homozygous rfl-1 (or19&s)
hermaphrodite worms raised to adulthood at six different temperatures ranging from 15°C
to 25°C (Figurel; see Materials and Methods). We chose 18°C (86.7% hatching) to screen
for enhancers of embryonic lethality and 23.5°C (12.7% hatching) to screen for
suppressors. These growth conditions optimized our ability to detect modifiers by
initially scoring qualitatively for changes in embryonic lethality, using stereomicroscopes
and 48-well agar plates with worm cultures (see Materials and Methods). At temperatures
higher than 23.5°C (24°C and above), most rfl-1 (or19&s) larvae matured into either
sterile or fertile adults with ruptured vulvae and produced very few or no progeny (data
not shown), precluding any use of higher temperatures to achieve a lower background
level of embryonic hatching for suppressor screening.
Quantifying Embryonic Viability to Identify Reproducible Enhancers and
Suppressors: To screen for modifiers, we used an RNAi feeding library of 16,757
bacterial strains, each capable of inducibly expressing double-stranded RNA
corresponding to C. elegans gene sequences [some E. coli produce dsRNAs that
correspond to two genes due to incorrect gene annotation] (KAMATH and AHRINGER
2003). We screened a total of 14,045 genes for suppression and 8,192 genes for
enhancement of rfl-1 (or19&s) embryonic lethality, after the mutant larvae matured into
adults while feeding on the dsRNA-expressing bacterial strains at the semi-permissive
temperatures. Our initial qualitative screen yielded 248 candidate enhancers and 388
candidate suppressors. We then consolidated and systematically re-screened each
29
100
wild-type
80
C
.....
- 60.....~
.....
;>
u
.....
s::
0 40~I-<
,L:J
S
P-l
~ 20
OL....L..---J..-...J..------'----'------'----'-------'---'-------'-----'.
15 16 17 18 19 20 21 22 23 24 25
Growth Temperature (OC)
Figure 1.- Temperature versus embryonic viability for rfl-l(or198ts) and wildtype
(N2). L1 larvae were raised to adulthood at the indicated temperatures on L4440
empty-vector control bacteria. Progeny were scored as hatched larvae or dead
embryos (n>200 except for 25°C where n=7 due to sterile phenotypes at this tempera-
ture; see Methods).
30
candidate enhancer and suppressor dsRNA using the same qualitative scoring method,
reducing the number of candidate enhancers and suppressors to I 0I and 104, respectively
(see O'Rourke et al 2007 for a description of the qualitative screening method).
To quantify the suppression and enhancement, we compared the embryonic
viability of broods from enhanced or suppressed young adults, and from control young
adults raised to adulthood on bacteria carrying the feeding RNAi vector without an insert
(hereafter referred to as empty vector control broods). Thirty-two of the candidate
enhancer genes, when reduced in function by RNAi, increased embryonic lethality in the
broods of rfl-1 (or198ts) mutants by at least 1.8 fold at 18°C, compared to empty vector
control broods (Figure 2A). To verify that depletion of these enhancer loci themselves did
not cause embryonic lethality, we depleted each of these 32 genes using feeding RNAi
and wild-type worms to quantify embryonic viability. None of the enhancer genes were
strongly required for embryonic viability under our conditions, with 5.7% embryonic
lethality being the most penetrant essential requirement we observed. We also tested each
of the 32 enhancers with rfl-1 (or198ts) mutants raised at 17°C, at which temperature 90%
(SD +/- 4%) of un-enhanced mutant embryos hatched. We found that fourteen of the 32
enhancers still increased embryonic lethality by two-fold or more at 1TC (Figure 2B).
We limited further analysis to these fourteen enhancers that reproducibly acted at both
semi-permissive temperatures (See Supplemental Table 1 Appendix Al for modifier gene
identities).
A similar quantitative brood analysis identified 21 suppressors that, when reduced
in function by RNAi, consistently increased the viability of embryos from
% Embryonic lethality (17°C)
W
......
>
......
o
o
ex>
o
0\
o
-1::>0
o
N
o
% Embryonic lethality (18°C)
I
'/' ' •...........
...•.......
" .
.J
.. ' ..
..
-
-
sel-l0
pha-l
abcf-3
ZK686.4
mtm-3
gpd-4
F32B6.3
cutl-8
vps-26
cnc-8
F25H9.7
T2003.3
fbl-l
T01G1.3
ZK185.2
ech-4
F38B2.4
T14E8.1
C07A9.5
Y43C5B.2
Y54GllA.ll
Y39G8C.2
C24010.1
fcd-2
T05B4.10
srh-292
srz-29
R09El0.5
K08F4.3
sqv-6
R05Al0.8
T01C3.2
L4440
to
o
o 0
ex>
o
0\
o
-1::>0
o
N
oo
~~10 .... ••
pha-l ....•....
abcf-3 .
ZK686.4 .•....•• +
mtm-3lJ .
gpd-4
F32B6.31-
cutl-8 .
vps-26'--
cnc-8
F25H9.7 I
T2003.3
fbl-l~ I
T01G1.3iii=
::iiH~
Y5:~~~g:~~
Y39G8C.2
C24dl0.l ~iiii~fcd-2,.
T05B4.1O
srh-292 F==-----,
srz-29 --<
R09El0.5;;;~:=1----K08F4.3
sqv-6
RO5A10.81:===:;::;::::;::::;-"
TO1C3.2 ~=""-"'"'
L4440
S"'OCD~
>-iae..o-"'I'j~ :::,.' ~ E.. 00'
....... g-~ s:.~
g" ....... CD O
>-i CD >-i 0 tv~ e.. (fQ 0-',~~CD~t'Ij~~~~e..
rJ). CD' ....... ~
tn· CD r '--' ~
'--' =l::o. ...... CD~!£ ~g80CD~~
>-i ::i t:l ::i 0
cr' _.;::l 0- H;~ a "d ,-...
>-i (D~ tt::J~
00 f""""!'- I "-" I
S' ~ ~ ...... >-.
o-_a......:lO'
o' :=;.. s n :::..~ '-<: >-i ~ 'C(tcr':::S~00
O
'-<:CD_ .....~ (fQ ........~~ ....... g. ::r- CD
rJ). (D~' ~ aEt~CDZO"'
::i ~ a > >-i
0- tv 0 .... '6
~ I ::l :::l ::ia S"' t:t ('D o'
o-p::So--
CD ~ rJ). ISS' CD~ ........ e; (t s:.
............. CDo-:::.
_ • ......:l ~ o-:=;.'
O~"""'CD'-<:\=i \.1 t:+"l"d •
qe;(;)'l'g"~. c .......
rJ). (fQ 0 m
CD ::r-::i ;.;
(fQr-'"O<
CD ,....., H; ~
:::s l W CD
CD 00' tv ~
rJ). e:' a CD~~~>-i(B cr' ::i CD
>-i e; 0- >-iCD r~ _. ~
v- 0--'
rJ). rJ).
CD S' g. CD(DO-CD 0-(')~. f""""!'-
....... a 0
CD g.
o-CD
32
hermaphrodites raised at 23.5°C by 1.8-fold or more, compared to worms raised on the
empty vector control E. coli (See Appendix for gene identities) . Depletion of thirteen of
the suppressors restored viability to over 38% hatching (3-fold over the control);
depletion of the strongest suppressor, csn-5, restored viability to 90% hatching.
Specificity of suppressors and enhancers: To test whether the modifiers we identified
specifically influence rfl-l function, or if depleting them can non-specifically influence
multiple conditionally mutant loci, we used four different temperature-sensitive
embryonic-lethal mutants that to our knowledge are not defective in functions related to
rfl-l or ubiquitin-mediated proteolysis: lit-l (or131ts), spn-4(or19lts), dhc-l (or195ts) and
spd-5(or213ts). The lit-l gene encodes a MAP Kinase that modulates Wnt signaling
(MENEGHINI et al. 1999); spn-4 encodes a protein with an RNA binding motif that
regulates cell fate patterning in the early embryo (GOMES et al. 2001); dhc-l encodes the
heavy chain of the minus-end-directed microtubule motor dynein (HAMILL et al. 2002);
and spd-5 encodes a coiled-coil protein required for centrosome maturation (HAMILL et
al. 2002). For each of these mutants we used growth temperatures that gave nearly
complete embryonic viability for testing enhancement, or nearly complete embryonic
lethality for testing suppression (O'ROURKE et al. 2007).We quantified the effects of
depleting modifier genes on embryonic viability with these four additional mutants and
compared the results to those obtained with rfl-l (or198ts) (Figure 3).
We found that while enhancers were often non-specific, most ofthe suppressors
were specific for rfl-l. Reducing the function of eight of the fourteen enhancers increased
rg
'"g'
-'""'
<7-
-<~(;"
3
5
~
rjl-1 (or198)23's"C
.... ""'UJ.b.VlCl'""'-lO)\,QO
00000000000
i , , • i , , iii
L4440 __ •
csn-5~~;;;~~~:':',csn-2mus-l07Rl0012.14
mnk-l
F25H5.5
F22B8.7
T24(4.3
(54010.1 C:=:::::JH
T2:J?s:~=
srl-63
B0491.4
Z(443.6
T2109.1
vig-l
M106.4
C35A5.1
T05H4.11
T04(9.1
gcy-20
L4440
csn-S
csn-2
mus-l07
Rl0012.14
mnk-l
F25H5.5
F22B8.7
T24(4.3
(54010.1
T26E3.6
elks-I
srl-63
80491.4
l(443.6
T2109.1[
vig-l I
M106.4
C35A5.1
T05H4.11
T04(9.1
gcy-20
spd-5(or213)23"( _ dhc-1(or195)23"( ~
.... ""'Ul~~O\""'-lo)\,QO .... ""'~~VlO\""'-lo)\,QO
00000000000 00000000000
Iii iii iii iii iii iii. i •
L4440
csn-S
csn-2
mus-l07
Rl0012.14
mnk-l
F25H5.5
F22B8.7
T24(4.3
(54010.1
T26E3.6
elks-7
srl-63
B0491.4
l(443.6
T2109.1
vig-l
M106.4
C35A5.1
T05H4.11
T04(9.1
gcy-20
lit-1 (01'131)23"(
-""'Ul~VlO\""'-lo)\,QO
00000000000
I , , , , , i , i ,
L4440
csn-S
csn-2
mus-l07
R10012.14
mnk-l
F25H5.5
F22B8.7
T24(4.3
(54010.1
T26E3.6
elks-I
sri-63
B0491.4
l(443.6
T2109.1
vlg-7
Ml06.4
C35A5.1
T05H4.11 L I
T04(9.1 -
gcy-20
spn-4(or191)23 "(
.... I'VUl~VlO\""'-lo)\,QO
00000000000
Iii iii iii i
spn-4(or191) 18"( lit-1(or131)20"( spd-5(or213) 15"( dhc-1(or195) 15"( rjl-1(or198)17"C
R09E10.5= I
F32B6.3 I
gpd-4
cutl-8
lK686.4!: I
mtm-3
T01C3.2
srh-292
T14E8.1
C24010.1
srh-292
T14E8.1
(24010.1
abcf-3
R09E10.5~
F32B6.3
gpd-4
cutl-B
lK686.4
mtm-3•••t--l
T01C3.2.....--I
sel-70L "pha-l H
R05A10.8
5RH-292
T14E8.1
(2:~~~::E
R09El0.5
F32B6.3
gpd-4
cutl-8
ZK686.4
mtm-3
T01C3.2
f
3
a
-'""'
'::?o
~.
~
!
~
Figure. 3- Specificity of modifiers for rfll{or198ts) embryonic lethality. Percent
embryonic viability (hatching) for suppression is shown on the left; percent embryonic
lethality for enhancement is shown on the right. Identity of conditionally lethal mutations
and growth temperatures are shown at the far left. Black bars indicate non-specific
modifiers; white bars indicate specific modifiers far left bar (L4440) is neg-ctrl RNAi.
w
w
34
embryonic lethality by at least 2-fold in one or more of the four other conditionally
mutant strains. Depletions ofthe remaining six enhancer loci specifically increased rfl-
1(or198ts) embryonic lethality by between 2.1 and 9.6 fold (21 and 96.4% lethality), but
reducing their function did not increase embryonic lethality by two-fold or more in any of
the four other conditional mutants we tested (Figure 3). In contrast, of the 21 reproducible
suppressor loci, 14 were specific for rfl-1 (or198ts): reducing their function failed to
suppress embryonic lethality by more than 2-fold in at least three of the four of unrelated
conditional mutants we tested (Figure 3). All of these 14 specific suppressors failed to
raise viability to 10% or more in any of the four unrelated mutants, while background
viabilities on empty vector control ranged from 1.2% for lit-1 (or131ts) as the lowest of
the four, to 3.7% for spn-4(or19lts) as the highest.
Two of the specific and most penetrant rfl-1 (or198ts) suppressors were csn-2 and
csn-5, which encode components of the COP-9 signalosome and have been shown
previously to suppress rfl-1 (or198ts) embryonic lethality when reduced in function
(PINTARD et al. 2003a). These were the only two signalosome components included in
the 14,045 gene set that we tested for suppression, confirming our ability to identify
functionally important suppressors.
Modifier Depletion and Cell Division Defects in ifl-l(or198ts) Mutant Embryos: We
next used DIC time lapse videomicroscopy to examine in live embryos the first mitotic
division in both suppressed and un-suppressed rfl-1 (or198ts) mutants. In un-suppressed
embryos from rfl-1 (or198ts) mutants grown at the fully restrictive temperature of25°C,
35
the failure to degrade MEI-l/katanin leads to defects in mitotic spindle orientation,
ectopic membrane furrows, and a failure to complete cytokinesis (KURZ et at. 2002). In
un-suppressed embryos, produced by rfl-1 (or198ts) mutant worms raised at 23°C and fed
bacteria carrying the empty vector, 54% of embryos failed to complete cytokinesis, 77%
had one or more ectopic cleavage furrows following cytokinesis, and the first mitotic
spindle was mis-oriented on the anterior-posterior axis in comparison to the wild type
(Figure 4). Depletion of the strongest suppressor, csn-5, almost completely rescued the
cytokinesis and ectopic furrowing defects: 0% and 10%, respectively, of the embryos
exhibited these defects, and the spindle orientation defect also was also suppressed
(Figure 4). Depletion ofthe specific suppressors mnk-1, mus-101, and RlOD12.l4
produced less complete but still substantial suppression of these defects in early stage
mutant embryos (Figure 4). We conclude that these specific suppressors influence the
same early embryonic processes that require rfl-1, and that the suppression of these cell
division defects may account at least in part for the increased viability of the suppressed
mutant embryos.
The synthetic embryonic lethality caused by depletion of specific enhancers in rfl-
1(or198ts) mutants raised at a semi-permissive temperature could result from further
compromising essential processes in the early embryo that require rfl-1, or from
deleterious interactions that occur later in development. We therefore used DIC time-
lapse videomicroscopy to examine the first mitotic division in enhanced and un-enhanced
mutant embryos, focusing on the two most penetrant and specific enhancer genes, set-10
and pha-1. At 17°C on empty vector RNAi-expressing bacteria, 0/15 un-enhanced rfl-
36
N
r-M..."....---------------,O
Rl0D12'14=::::~~_.n=10mus-101 n-10
~ mnk-1 n-9
0:: csn-S.n=10
L4440 n 13
o 10 20 30 40 50 60 70 80
Percent of embryos with Ectopic Furrows
Rl0D12.14-===~~ n = 10
mus-101_ n = 10
~ mnk-1 n=9
rr:
c$O-5 n=10
L4440 n = 13
o 10 20 30 40 50 60
Percent of embryos with Cytokinesis Failure
NEB Cytokinesis
Figure 4.- Suppression of early embryonic cell division defects in rjl-l (or198ts) mutants raised
at 20"C and then shifted to 23°C for 7-12 hours prior to imaging (see Materials and Methods). (A-
L) show frames from time-lapse DIC videomicrographs at nuclear envelope breakdown (A, E, I),
during cytokinesis one minute after fIrst appearance of a cleavage furrow (B, F, J), and at the 2-
cell stage after the completion of cytokinesis (C, G, K). Black arrowheads indicate centrosome
position; white arrowheads indicate ectopic membrane furrows; white arrow indicates nuclei of
AB cell and P I cell advancing toward each other after cytokinesis failure; black arrow indicates
regression of cytokinetic furrow. Temperature-controlled stage prevented use ofoptimal DIC
optics (see Materials and Methods) (M) Quantillcation of ectopic cleavage furrows in suppressed
and controls embryos. (N) Quantitation of cytokinesis failure in suppressor-RNAi treated and
control RNAi treated embryos. Number of embryos scored are indicated for each condition in
(M) and (N). (0) Angle of mitotic spindle at NEB and at cytokinesis (1 minute following
appearance ofcleavage furrow). Hash marks indicate individua Idata points and shading indicates
mean angle of all data points.
37
1(or198ts) embryos exhibited cytokinesis failures, while 2/15 had ectopic cleavage
furrows. At 18°C, 2/15 empty vector RNAi-fed embryos exhibited cytokinesis failure and
5/15 had ectopic furrows. While sel-10(RNAi) and pha-l (RNAi) both dramatically
increase the rate of lethality at these temperatures, we observed no increase in cytokinesis
failures, ectopic furrowing, or spindle orientation defects at either 17°C or 18°C (Table 1).
This lack of enhancement of early cell division defects suggests that the synthetic lethal
interaction(s) may occur at a later stage in embryonic development, or are not detected by
our methods.
To ask whether rfl-l has functional requirements later in embryogenesis, beyond
the first few cell divisions, we performed temperature up-shift experiments with rfl-
1(or198ts) embryos, following the completion of early cell divisions at the permissive
temperature of 15°C. After shifting 16-cell and later stage embryos to 26°C, we found
that 18/42 embryos (48.2%) failed to hatch, while in a control experiment with no
temperature up-shift only 3/51 embryos (5.8%) failed to hatch, indicating that rfl-l does
have additional essential requirements later in embryonic development. We examined the
terminal phenotypes of the un-hatched embryos using DIC microscopy and found that
16/18 embryos arrested after elongation to or beyond the 3 fold stage. We also used DIC
microscopy to examine enhanced rfl-l mutant embryos produced at 18°C after sel-l 0
depletion and found that 25/28 (89%) of these enhanced mutant embryos failed to hatch,
and many of the unhatched embryos appeared to arrest after little if any elongation (data
not shown), a more severe phenotype than we observed after up-shifts of un-enhanced rfl-
1(or198ts) embryos to 26°C (see above). Other rfl-l(or198ts) embryos enhanced by sel-
38
TABLE 1
Modifier depletion does not enhancer early embryonic cell division defects in ifl-
1(or198ts) mutants.
Genotype # embryos # embryos with cytokinesis
with ectopic failure
furrows
rjl-1(or198);empty vector RNAi 17°C 2/15 0/15
rjl-(or198); sel-10(RNAi) 17°C 2/11 0/11
2/11 0/11
rjl-1 (or198);pha-1 (RNAi) 17°C
rjl-1 (or198); empty vector RNAi 18°C 5/15 2/15
rjl-(or198); sel-10(RNAi) 18°C 5/13 0/13
rjl-1(or198);pha-1(RNAi) 18°C 3/13 1/13
10 depletion arrested after variable amount of elongation (data not shown). We conclude
that the synthetic lethal interactions observed after enhancer depletion could result from
interactions that occur after the early embryonic cell division processes known to require
rjl-1. However, we have not determined precisely where or when in embryogenesis the
synthetic lethal interactions occur.
Suppressor and enhancer protein localization overlap with RFL-l: To gain further
insight into how the specific modifiers might influence rjl-1 dependent processes, we first
constructed N-terminal GFP fusions to rjl-1 and to five of the specific suppressors, all
driven by the maternal pie-1 promoter, and isolated transgenic lines for all six fusions
(see Methods). Previous immunocytochemistry studies of the neddylation pathway
39
components NED-8 and CSN-5 documented nuclear and cytoplasmic localization in
fixed embryos, with a pronounced nuclear enrichment (KURZ et al. 2002; PINTARD et al.
2003a; PINTARD et al. 2003b). Consistent with these previous studies ofNedd8 pathway
components, we observed a similar nuclear enrichment of the GFP::RFL-l fusion protein
in live embryos, using spinning disk confocal microscopy (Figure 5; see Methods).
Fusion constructs for F25H5.5 (an ortholog of the human Protein CLASPIN), F22B8.7
(an uncharacterized conserved iron-sulfur domain-containing protein), T26E3.6 (related
to Fibrillin), and MNK-l (a conserved kinase) all displayed nuclear enrichment in early
embryonic cells. GFP::RlOD12.14 (a conserved protein of unknown function) localized
more exclusively to the cytoplasm, appearing to be excluded from nuclear and mitotic
spindle regions. In addition, GFP::RlOD12.14 was cortically enriched near cytokinetic
furrows, particularly during the division of the anterior-most two-cell stage blastomere,
and at cell boundaries in oocytes (Figure 5 and data not shown). While these are very
general localization patterns, most of the suppressor proteins exhibit localization patterns
similar to those observed for RFL-l, NED-8 and CSN-5, consistent with the possibility
that these suppressor proteins influence the neddylation pathway or the regulation of
MEI-l/katanin in early embryonic cells.
We also examined the expression and localization of GFP fusions to the proteins
encoded by the two most specific enhancers, SEL-IO (an F-box substrate adaptor for
SCF-type E3 ligases) and C24DlO.l (an uncharacterized protein tyrosine phosphatase),
using recombineering to construct GFP translational fusions regulated by native promoter
and other non-coding sequences (see methods). We did not detect any expression of
40
Pronuclear Metaphase Telophase 2-Cell
I
. ,~
,
. "f 'J
.~. t:;~ ,
-' .~"
., '
. . ., ,
" .
, .
• '. J' •
. ' .
: " "' ... ~< ~." ~
f ~ ,;:-
'. ,:)~~~\(~»; ,
< ~.'
"""
.....
N
.....
o
o
;:2
Figure 5.- Expression ofGFP fusion to suppressor encoded proteins in early embryos
from transgenic strains. Frames fi'om single optical sections at indicated cell cycle stage
fro m sp inning-d isk confocal time-lapse videomicrograp hs (see Materials and Methods).
Scale bar equals 10 flM.
41
GFP::SEL-10 in early embryos, but we did detect cytoplasmic expression in some cells
beginning at about the 50-cell stage (Figure 6A). We detected higher levels of
cytoplasmic expression beginning at about the bean stage (Figure 6B), after the
completion of most embryonic cell divisions, in many cells throughout the embryo. In
larvae and adults, we observed expression in head and tail neurons and in unidentified
cells along the entire length of the body (Figure 6). For the fusion GFP::C24D10.l, we
again did not detect any expression in early embryos but first detected expression
beginning at approximately the bean stage of embryogenesis, predominantly in nuclei and
in many cells throughout the embryo (Figure 6 D-E). In larvae and adults, we observed
GFP::C24D10.1 in the nuclei of many cells throughout the head.
The lack of early embryonic expression for the two most penetrant enhancers
could be due to transgene silencing in the maternal germline, a frequent outcome for
maternally expressed genes in transgenic C. elegans strains (KELLY and FIRE 1998).
However, we did detect presumably zygotic expression of the GFP fusions to these
proteins in later stage embryos, and we wanted to compare these later expression patterns
to RFL-1. We therefore used recombineering to produce an N-terminal-tagged
tdTomato::RFL-1 fusion driven by the rfl-l promoter and generated a transgenic line that
expresses this fusion (see Materials and Methods). We again did not observe any early
embryonic expression, presumably due to germline silencing. However, we did detect
strong cytoplasmic expression in larval stages and in adults, in both head and tail
neurons, and in vulval epithelial cells and intestinal cells (Figure 6), consistent with
previous studies of a rfl-l::GFP promoter fusion as a transcriptional reporter (HUNT-
42
Figure 6.- Spinning disk confocal projected Z-stack images ofGFP and tdTomato
fusion proteins (see Materials and Methods).GFP::SEL-IO expression (A-C, G, H). (A)
~50-cell stage (B) bean stage (C) comma stage (G) expression in head ofadult (H)
Expression in tail neurons. GFP::C24DIO.I expression (D-F, I). Bean stage (D); comma
stage (E); 1.5-fold stage (F); head ofadult around pharynx (1). tdTomato::RFL-1 (J-L).
(J) Tail neurons and intestine in adult stage; (K) Vulval VulB2 cells of young adult; (L)
larval head. Anterior is to the left; scale bars equal 10~.
43
NEWBURY et al. 2007). To summarize, although the two strongest specific rjl-l (or198ts)
enhancers are expressed in later stage embryos, we did not detect any RFL-l expression
in later stage embryos. For this reason, and because we did not detect any maternal
expression of the two enhancer GFP fusions, we do not know in which cells RFL-l might
interact with SEL-IO or C24DIO.l during embryogenesis, assuming such interactions are
responsible for the enhanced embryonic lethality we observe. It is possible that such
interactions do occur in later stage embryos but involve undetected low levels of RFL-l,
or maternal expression of the two enhancers. Finally, while functional requirements for
RFL-l in larvae and adults remain uncharacterized, the expression of RFL-l in vulval
epithelial cells may relate to the frequent rupture at the vulva in young adult
hermaphrodites raised at restrictive temperatures (data not shown).
To further investigate the relationship of the RFL-l modifiers we have identified,
we asked if depletion of the suppressor gene products can alter the localization of
GFP::RFL-l, and if depletion of rjl-l or other neddylation pathway components can
change the localization of the GFP suppressor protein fusions in early embryonic cells.
We did not detect changes in localization for any of the suppressor fusions following rjl-
l, nedd-8 and ula-l depletions (data not shown). Similarly no change in GFP: :RFL-l
localization was observed upon depletion of the suppressor genes mus-lOl, RlOD12.14,
mnk-l, F25H5.5, T26E3.6 or F22B8.7 (data not shown). We also did not observe any
change in early embryonic GFP::RFL-l localization upon depletion of sel-lO, pha-l, or
C24DIO.l, a result consistent with our finding that sel-lO and pha-l depletion do not
enhance the cellular phenotypes in rjl-l mutants.
44
Finally, we also examined GFP::RFL-l expression in transgenic embryos after
depleting the neddylation pathway components ula-l, nedd-8, and ubc-12 (Figure 7). We
did not observe changes in RFL-llocalization after depletion either nedd-8 or ubc-12, but
we did observe a loss of GFP::RFL-l nuclear enrichment following ula-l depletion.
ULA-l and RFL-lIUBA-3 form the El activating enzyme complex for the Nedd8
conjugation pathway, with RFL-l being the catalytic AAA-ATPase subunit (JONES and
CANDIDO 2000). A comparison of pixel intensities showed a nuclear/cytoplasmic ratio of
1.01 after ula-l depletion, compared to 1.49 in wild-type embryos (n = 6 embryos for
each analysis and then averaged; see Material and Methods) Using western blots, we
detected similar levels ofGFP::RFL-l in extracts from ULA-l depleted worms and wild-
type worms (data not shown), suggesting that ULA-l is required for RFL-l nuclear
enrichment independent of any changes in protein levels. To our knowledge, such a
requirement for ULA-l orthologs has not been reported in other organisms.
45
GFP::RFL-l----------
L444(XRNAi) ula-4(RNAi) ned-8(RNAi) ubc-12(RNAi)
Figure 7.- Nuclear localization ofGFP::RFL-l is lost in ula-1(RNAi) but not in other
neddylation pathway defective backgrounds.
DISCUSSION
To identify factors that influence the regulation of cell division in the early C.
elegans embryo, we used a high throughput RNAi screen to identify suppressors and
enhancers of the embryonic lethality associated with or1981s, a temperature-sensitive,
embryonic-lethal mutation in the C. elegans gene rjl-1, which encodes a component of
the ubiquitin-like Nedd8 protein conjugation pathway. Using ts mutations in other
essential loci, we tested the modifiers to detemline if they were specific for rjl-1(or198ts)
embryonic lethality. We found that many of the enhancers and suppressors were non-
specific, with specific suppressors being more common than specific enhancers.
Depletion of the rjl-1-specific suppressors partially rescued the embryonic cell division
defects associated with loss ofrjl-1 function, and similar sub-cellular distributions ofthe
suppressor proteins and RFL-l are consistent with the suppressors having RFL-l-related
46
roles in early embryonic cell division. We also identified two highly penetrant and
specific enhancers of rjl-l (or198ts) embryonic lethality, but their depletion did not
detectably enhance early embryonic cell division defects in mutant embryos. We
speculate that rjl-l may have roles later in development that can explain the synthetic
lethality that results when these enhancers are depleted in rjl-l (or198ts) mutants raised at
a semi-permissive temperature.
Synthetic screening with RNAi and temperature-sensitive, embryonic-lethal C.
elegans mutants--controlling for specificity: While some large-scale synthetic screens
using RNAi and temperature-sensitive C. etegans mutants have addressed the issue of
modifier specificity (FRASER 2004; LEHNER et at. 2006; O'ROURKE et at. 2007),
relatively few such screens have been reported thus far. Moreover, this study represents
the first time that a systematic and quantitative analysis of specificity has been reported
for both enhancers and suppressors of a single embryonic-lethal C. etegans mutant.
In our analysis of the modifiers, we found that many of the enhancers were non-
specific, while more suppressors were specific for rjl-l. Of the 21 suppressors 71 % were
specific: their depletion did not restore embryonic viability to other conditionally
embryonic-lethal mutants grown at semi-permissive temperatures. In contrast, only 43%
of enhancers were specific, and only 14% (2 of 14) strongly enhanced embryonic
lethality. Enhancers we identified in modifier screens with other conditional mutants also
exhibit, with very few exceptions, a high degree of non-specificity when tested with
multiple conditionally lethal mutants (M.D. and S.O., unpublished data). We conclude
47
that when using RNAi to reduce gene function in temperature-sensitive, embryonic-lethal
C. elegans mutant backgrounds, synthetic embryonic lethality most often results from
additive and unrelated defects and probably does not result from the two genes
influencing the same biological process or pathway. While genes that enhance embryonic
lethality when depleted in more than one mutant background could represent functional
links among the genes mutated in the different backgrounds, we suspect that most such
synthetic-lethal interactions instead involve un-related defects.
Synthetic lethal screens in budding yeast have been powerful tools for the
discovery of new gene functions (001 et al. 2006; TONG et al. 2004), and it is interesting
to compare the specificity observed in synthetic lethal screens done in C. elegans with the
specificity observed in synthetic knockout screens done in budding yeast. For example,
one yeast study analyzed 132 nonessential genes (TONG et al. 2004). Each of these 132
viable knockout mutants was mated with ~4700 additional viable knockout mutants to
produce double mutants. A total of~4000 synthetic lethal interactions were identified,
with an average of 34 interactions per mutant. Of the interactions found, 27% were with
pairs of genes known to be in the same or related genetic pathways. These findings
suggest a high degree of specificity for synthetic lethal interactions in yeast, and that the
interactions reflect participation in a common process. By contrast, one study in C.
elegans found that over half of the genes on chromosome III, when reduced in function
by feeding RNAi, significantly enhanced the embryonic lethality of a hypomorphic
mutation in the transcription factor gene dpl-I (FRASER 2004). Similarly, we found that
many of the strong enhancers, when depleted by RNAi, enhanced embryonic lethality
48
with multiple conditional mutants. This high degree of non-specificity compared to the
synthetic lethal screens in budding yeast could reflect the added genetic complexity of the
cellular and developmental processes in a multicellular organism. Alternatively, the
different degrees of specificity may simply reflect the different kinds of mutant alleles
used for screening. The yeast synthetic-lethal screens used double mutants made with
viable deletion alleles. In contrast, the synthetic screens in C. elegans have used partial
inactivation of essential genes, usually by growing temperature-sensitive mutants at
intermediate temperatures to sensitize genetic backgrounds, and RNAi to deplete the
expression of other genes that mayor may not themselves be essential. Perhaps
temperature-sensitive mutants grown at just-viable temperatures are particularly
vulnerable to non-specific, synthetic lethality caused by the disruption of unrelated
processes. It would be interesting to use genome-wide feeding RNAi screens to detect
synthetic lethality in C. elegans strains that are homozygous for deletion mutations in
non-essential genes. Conversely, it would be interesting to know if temperature-sensitive
mutations in essential budding yeast genes are also prone to high levels of non-specific
enhancement and suppression when other genes are reduced in function.
While non-specific enhancement of embryonic lethality is especially common,
suppression of embryonic lethality by RNAi knockdown of other C. elegans loci also is
frequently non-specific. In a genome-wide RNAi screen for suppressors of a temperature-
sensitive dynein heavy chain mutant, dhc-1 (or195ts), 49 genes were identified that, when
depleted by feeding RNA, restored dhc-1 (or195ts) embryonic viability by 3-fold or more
(O'ROURKE et al. 2007). However, depletion of 57% of the dhc-1 (or195ts) suppressors
49
also significantly suppressed embryonic lethality for at least one of two other mutants
with temperature-sensitive mutations in essential loci unrelated in function to dhc-l. As
we report here, 29% of the rfl-l (or198ts) suppressors were similarly non-specific. For at
least some of the non-specific dhc-l (or195ts) suppressor genes, their depletion has been
reported by others to increase glycerol production, which may non-specifically stabilize
temperature-sensitive proteins and thereby increase embryonic viability at serni-
permissive temperatures (LAMITINA et al. 2006; O'ROURKE et at. 2007). Clearly, it is
important to carefully assess the specificity of both enhancer and suppressor interactions
that influence the degree of lethality associated with temperature-sensitive mutations in
essential C. elegans genes. Many interactions are highly non-specific and presumably
will not provide mechanistic insight into the conditionally defective biological process.
Known roles for specific modifier genes: One ofthe strongest specific suppressors,
mus-l 01, encodes a widely conserved 1227 amino acid protein containing six copies of
the BRCAI carboxyl-terminal (BRCT) repeat (HOLWAY et al. 2005; YAMAMOTO et al.
2000). The mus-10l gene is essential but feeding RNAi results in only partially penetrant
embryonic lethality (MAEDA et at. 2001). BRCT domains are commonly found in
proteins involved in DNA metabolism, and have been implicated in mediating protein-
protein interactions between other BRCT domain-containing proteins (MANKE et al.
2003; Yu et al. 2003). Some proteins with BRCT repeats, including BRCAl, have roles
in mitosis as well as more established roles in DNA replication and repair. For example,
BRCAI binds to tubulin and localizes to centrosomes and spindle microtubules (Hsu and
White, 1998), and high levels ofBRCAI are maintained throughout mitosis, while the
50
protein is ubiquitinated and degraded during Gland S phase (Choudhury et ai., 2004).
Furthermore, in both mammalian and Xenopus cells, BRCA1 is required for spindle pole
assembly and the centrosomal accumulation of TPX2 (JOUKOV et al. 2006). Perhaps the
BRCA repeat protein encoded mus-l0l may also influence cell division functions such
that it can restore viability when depleted in rjl-l mutants.
The other rjl-l suppressor with a possible link from other studies to cell division
is R10D12.14. This gene is at least partially essential (PIANO et al. 2002), but its specific
cellular requirements remain unknown. R10D12.14 encodes a protein with a GYF
domain (poly-proline interaction motif) and was identified as a binding partner of Dynein
Light Chain (DLC-1) in a large scale two-hybrid interaction screen of metazoan-specific
genes from C. elegans; DLC-1 was the only binding partner ofR10D12.14 identified in
this study (LI et al. 2004). While the link to dynein might reflect roles in cell division, the
GFP::R10D12.14 fusion protein we examined was largely excluded from nuclei and
mitotic spindles and was mostly cytoplasmic, with some cortical enrichment at
cytokinesis furrows and in oocytes.
The two strongest specific enhancer genes were sel-l0 and pha-l. The sel-l0 gene
encodes an F-box protein and has been implicated as a substrate adaptor in E3 ligases that
control the ubiquitin-mediated degradation ofLIN-12/J\Jotch, SEL-12/Presenilin, and the
sex-determining proteins FEM-1 and FEM-3 (HUBBARD et al. 1997; JAGER et al. 2004;
Wu et al. 2001). There are no identified requirements for Cullin neddylation in the
ubiquitin-mediated degradation of SEL-1 0 targets, but our finding of a strong and highly
51
specific enhancement of embryonic lethality raises this possibility. The pha-I gene
encodes a novel protein that has been shown to function redundantly with class B
SynMuv genes, such as lin-35/Rb and ejl-I, to influence pharyngeal morphogenesis (FAY
et al. 2004). Furthermore, reducing the function of both pha-I and anyone offour
ubiquitin ligases -ubc-18, C27A12.6, C27A12.7, ari-I-results in pharyngeal
morphogenesis defects and partially penetrant early larval lethality (QIU and FAY 2006).
ARI-1 has been shown to interact with CSN-5 by yeast two-hybrid screening, and
inactivation of csn-5 also enhances pharyngeal defects in some mutant backgrounds (D.
Fay, personal communication). Our results suggest that neddylation may be important for
proper proteolytic regulation ofpha-I and/or lin-35/Rb dependent processes.
Identifying requirements for non-essential genes: While mutational studies and
genome-wide RNAi screens in C. elegans have identified thousands of genes that are
either essential or have visible requirements, roughly 70% ofthe known and predicted
genes in the C. elegans still have no known requirements (KEMPHUES 2005) even
though at least halfhave homologs in other animal phyla (LANDER et al. 2001).
Understanding the function of conserved but non-essential genes is an important frontier
of genetic research in model organisms, as many such genes are widely conserved and
could be relevant to human disease processes. Moreover, non-essential genes that
influence essential processes may prove to be valuable drug targets: altering their
function may allow for modification of an essential process without causing deleterious
side effects that might result from targeting a more pleiotropic and essential disease gene.
In our screen for modifiers of the essential C. elegans gene rjl-I, 11 of the 16 specific
52
suppressors and all 6 of the specific enhancers we identified appear to be non-essential.
Thus when appropriately controlled for specificity, modifier screens that use RNAi and
conditional mutations in essential C. elegans genes may prove useful in identifying roles
for non-essential but conserved genes.
53
CHAPTER IV
NOVEL REQUIREMENTS FOR THE GENES Y119CIA.l AND DRH-3 IN
WNTIMAPK PATHWAYS AND ENDODERM SPECIFICATION IN C. ELEGANS
The screening procedure utilized to identify genetic modifiers of lit-1 (or131ts)
was developed primarily by Sean O'Rourke, Jose Gomez and myself. The identification
of lit-1 (or13lts) modifier genes and subsequent characterization analysis described in this
chapter, was performed entirely myself.
INTRODUCTION
Wnt signal transduction pathways utilize highly conserved molecular components
and mechanisms that play fundamental roles in embryonic development, tissue
regeneration, cell polarity and cancer (CADIGAN and NUSSE 1997). In C. elegans, both a
Wnt signaling pathway and a MAPK pathway are necessary for the posterior versus
anterior sister cell-fate specification in developing embryos (MENEGHINI et al. 1999).
These pathways are first utilized in the four-cell stage embryo where a MOM-2/Wnt
signal from the Pz blastomere signals anteriorly to the adjacent EMS cell, ultimately
leading to its polarization and subsequent asymmetric cell division. The daughters of this
cell division, MS and E, go on to become the mesoderm and endoderm, respectively
(ROCHELEAU et al. 1997; ROCHELEAU et al. 1999; THORPE et al. 1997).
54
The Wnt and MAPK signaling pathways required for EMS cell polarization have
been studied extensively and genetic and molecular characterization have identified many
components of each pathway. One of the first components to be identified was the gene
pop-I, a member of the TCF/LEF family of transcription factors. In pop-l mutants both
daughters of the EMS cell adopt the MS cell fate. In wildtype embryos, lower levels of
nuclear localized POP-1 are found in E (posterior cell) relative to MS. This relative
difference does not appear to be due to POP-1 degradation in the E cell, but appears to be
the result of a redistribution of nuclear POP-1 to cytoplasmic POP-1 in the E blastomere
(MADURO et al. 2002). Together, these results support the model that unlike other
TCF/LEF proteins in other Wnt pathways, that serve to activate transcription of target
genes, POP-1 acts as a transcriptional repressor ofMS cell fate genes. However, a recent
study has revealed that remaining POP-1 protein in E cell nuclei acts as a transcriptional
activator when bound to the ~-catenin homolog SYS-1 (KIDD et al. 2005). Many other
components of the Wnt and MAPK pathways were first identified as being required for
the E cell adopting the endoderm fate, and were later shown to be required for the lower
levels of nuclear POP-1 in the E cell. These genes were named for their shared mutant
phenotypes: MOM (more-of-mesoderm) and LIT (loss-of-intestine).
Wnt signaling is induced by Wnt/MOM-2 (ROCHELEAU et al. 1997; THORPE et al.
1997) binding the receptor MOM-5/Frizzled (ROCHELEAU et al. 1997; THORPE et al.
1997) on the EMS cell which activates the downstream cytoplasmic components SGG-
l/GSK-3 and APR-1/APC. In canonical Wnt signaling, GSK-3 and APC are components
of the ~-catenin destruction complex (XING et al. 2003) that are degraded in response to
55
the Wnt signal. The degradation of the ~-catenin destruction complex allows for
cytoplasmic accumulation of ~-cateninand its subsequent translocation into the nucleus
where it binds to and activates the TCF/LEF transcription factor. Curiously in C. elegans,
EMS cell polarization requires the presence and positive activity of both SGG1 and APR-
1 for WRM-l/ ~-cateninmediated negative regulation ofPOP-l in the E cell (YOST et al.
1996). It remains unclear what specific roles SGG-I and APR-I have in EMS cell
polarity establishment.
The Wnt pathway converges with the MAPK pathway which includes the
MAPKKK MOM-4 (SHIN et al. 1999), a homologue of vertebrate transforming-growth-
factor TAK1, and the NEMO-like kinase LIT-I (MENEGHINI et al. 1999). MOM-4 is
required for LIT-1 kinase activity and functions upstream of LIT-I. LIT-1 forms a
complex with ~-catenin/WRM-I. In vitro experiments have shown that LIT-I can
phosphorylate POP-I, and WRM-I mediated activation of LIT-I is required for
phosphorylation of POP-l in vivo (ROCHELEAU et al. 1999). The WRM-l/LIT-1 complex
translocates into the nucleus of the E cell and phosphorylates POP-l leading to its export
from the nucleus. Consistent with this function, LIT-1 is emiched in the posterior nuclei
(Lo et al. 2004). Recently, the conserved 14-3-3 protein PAR-5 was shown to be required
for POP-I asymmetry and likely has a direct role in promoting the export of
phosphorylated POP-I from the nucleus (Lo et al. 2004). Proteins containing 14-3-3
domains previously had been shown to regulate the nuclear export of their binding
partners (LOPEZ-GIRONA et al. 1999). One other factor that has been shown to be required
for POP-I nuclear export is ZK742.1 which is the homolog of the vertebrate exportin
56
CRM-l. RNAi mediated knock-down of crm-1 inhibits nuclear export of POP-l from the
E cell nucleus and also causes a failure in endoderm development, in a strain containing
an N-terminal, GFP tagged POP-l (La et ai. 2004). However, the loss ofGFP::POP-l
nuclear asymmetry only occurs in 40% of AP sisters in the EMS lineage, indicating that
RNAi does not cause complete knock-down of crm-1, or that other factors are sufficient
for POP-l nuclear export. It is not known what causes the asymmetry of LIT-l although
knockdown of both Wnt and MAPK signaling pathways causes a loss of LIT-l
enrichment in posterior nuclei. There are likely other factors required for recognition
and/or transport of phosphorylated POP-l and LIT-1 between the nucleus and cytoplasm.
To identify additional factors that influence POP-l activity and localization and
Wnt signaling dependent cell polarity in C. eiegans, we have used RNA interference
(RNAi) to reduce gene functions in the temperature-sensitive lit-1 mutant or131ts (see
chapter 2 and 3 for information of RNAi, screening methods, and discussion of sensitized
genetic backgrounds). Here we report our identification of two genes that when reduced
in function by RNAi, modify the amount of embryonic lethality in lit-1 (or131ts) mutants.
We go on to describe the initial characterization of these two modifiers that display both
a very high degree of penetrance and genetic specificity for lit-1 (or131ts) mutants. One
of the modifiers, Y119C1A.l, is a protein containing two conserved domains, and when
depleted with RNAi causes a 188.6-fold (0.5% with negative control and 94.3% with
Y119CIA.1 RNAi at 17.5°C) increase in lit-1 (or13lts) embryonic lethality. Remarkably,
depletion of YI19CIA.l has no loss-of-function phenotype of its own and fails to
enhance lethality in five other ts mutants, each defective in other essential embryonic
57
processes. We also describe the identification and characterization of the conserved and
essential gene drh-3, that produces strong and highly specific suppression of lit-
I (orI3lts) embryonic lethality when depleted with RNAi.
MATERIALS AND METHODS
Screening procedure and quantitative scoring: Techniques used for screening and
scoring are identical to those described in Chapter III methods section.
Dark field microscopy: Visualization of intestinal granules was done using an Ziess
Axioskop (Microscope) and a Plan-Apochromat 63XIl,4 oil immersion objective.
Molecular biology: To construct the pei-I driven N-terminal GFP fusion construct of
YI19CIA.I we first used Pfu Turbo polymerase (Stratagene) and a eDNA library
(Invitrogen), and amplified the gene using the following primers containing spe-I and
asc-I restriction sites (lowercase): 5'-ggtactagtATG CCT CAT CAA CCA GTG CT-3';
5'- gtgtggcgcgccTTA GGC AGT GGA ATT AGC TGT G-3'. PCRproducts were
subsequently ligated into pGEM-T or pGEM-T-easy shuttle vectors (Promega). Inserted
genes were sequenced in the University of Oregon sequencing facility prior to cleavage
and ligation into pS026 [pS026 described in (O'ROURKE et al. 2007)]. Transformation of
constructs using particle bombardment is described in Chapter III methods.
Spinning Disk Confocal Microscopy: Equipment and techniques used for imaging
GFP::YI19C IA.I are the same as described in Chapter III methods section.
58
RESULTS
A Sensitized Genetic Background: To identify semi-pennissive temperatures that
optimize our ability to identify enhancers and suppressors of lit-1 (or131ts) embryonic
lethality, we examined the viability of embryos produced by homozygous lit-1 (or131ts)
hermaphrodite worms raised to adulthood at five different temperatures ranging from
15°C to 25°C. We chose 20°C (24% hatching N>200 progeny) and 23°C (1.04% hatching
N>200 progeny) to screen enhancers and suppressors of embryonic lethality,
respectively. These growth conditions optimized our ability to detect modifiers by
initially scoring qualitatively for changes in embryonic lethality, using stereomicroscopes
and 48-well agar plates with worm cultures. For later analysis and rescreening of lit-1
enhancers we used a less restrictive temperature of 17.5°C (99.5% hatching) for assessing
enhancement of embryonic lethality as this allowed for even greater sensitivity and
reproducibility of results.
Identification of lit-l(or131ts) genetic modifiers: Using the RNAi feeding library we
screened a total of 12,874 genes for enhancement and 15,716 genes for suppression of lit-
1(or131ts) embryonic lethality. Our initial quantitative screen yielded 328 candidate
enhancers and 239 candidate suppressors. We consolidated and systematically rescreened
each candidate enhancer and suppressor using the same qualitative scoring method,
59
reducing the number of candidate enhancers and suppressors to 89 and 31, respectively
(see Chapter 2 for a description of the qualitative screening method).
To quantify suppression, we compared the embryonic viability of broods from
suppressed young adults, and from control RNAi treated young adults raised to adulthood
on bacteria carrying the feeding RNAi vector without an insert (same method as in
Chapter 3). From this analysis, five of the candidate suppressors, increased embryonic
viability in the broods of lit-1 (or131ts) mutants at 23°C by at least 6-fold over
background (quantitative data shown in Figure 1).
The relatively large number of genetic enhancer candidates remaining after
qualitative screening, and the high degree of non-specificity generally observed with
enhancers of ts mutants, led us to perform an additional round of screening to aid in
identifying the most penetrant and specific candidate modifiers before proceeding with
quantitative analysis (see methods). Following this additional screening procedure we
identified 12 genes that when reduced in function by RNAi enhanced lethality in lit-
1(or13lts) mutants that by qualitative analysis did not enhance two unrelated ts mutants
dhc-1 and spd-5 (data not shown). Only one of these RNAi clones, YI19CIA.l a gene
containing a RanBP-Type 2 domain and a DNA methylase domain, produced a dramatic
increase in lethality observed in all rounds of qualitative screening, while the other 11
clones produced only mild enhancement (less than ~2-fold increase). Following
sequencing the vectors to verify the identity of the RNAi clone, we quantified the
enhancement caused by RNAi depletion ofYl19CIA.l in lit-1(or131ts) mutants and
found that at 17.5°C lethality is increased to 94.3% (n=539) from 0.5% (n=985) on
60
empty-vector RNAi. At temperatures ~20°C adult fit-1 (or13its) worms become sterile
when depleted of YI19CIA.1.
Specificity analysis yields two highly specific lit-l(or13lts) modifiers: Modifiers of
embryonic lethality in ts mutant backgrounds often function in a non-specific manner, as
they can enhance or suppress multiple unrelated mutant backgrounds (See Chapter III and
(O'ROURKE et al. 2007). We performed RNAi treatment of each ofthe five suppressors
and the fit-1 (orl3its) enhancer YI19CIA.I with five other ts mutants: spn-4(or19its),
dhc-1(or195ts), spd-5(or213ts), rjl-1(or198ts) and mel-26(or543ts). We found that the
enhancer YI19CIA.I and a suppressor drh-3, a gene containing a dead-box helicase, and
has been shown to regulate RNAi induced gene silencing in the germline, are both
completely specific modifiers as they fail to significantly modify lethality in any of the
other five mutants tested (Figure 2). This very high genetic specificity and penetrance,
suggests that drh-3 and YI19CIA.I have roles that directly influence Wnt signaling
and/or cell polarity in the C. elegans embryo.
Allele specificity and genetic interactions with mom-4: Genetic modifiers of fit-
1(or13its) may function to influence Wnt or MAPK signaling, or they may act in a way
that compensates for the specific molecular defect caused by the or131ts mutation. We
therefore, used two additional mutant alleles of lit-1 to evaluate the effect of both drh-3
and Y119CIA.I depletion on embryonic lethality. We observed strong enhancement of
both fit-1 (ne199its) and lit-1 (or393ts) embryonic lethality with RNAi depletion of
61
14440gtl-lW03G9.4osr-ldrh-3C50D2.1
o
70 -,--~-------------------------..,
60
10
Figure 1.- Suppression of lit-1 (or131ts) lethality by RNAi.
62
Yl19ClA.l (Figure 2). We also observed significant suppression of embryonic lethality
with RNAi depletion of drh-3 (Figure 2). These results are consistent with both modifiers
somehow influencing lit-l related processes, and not simply compensating for an
unstable or miss-folded LIT-l protein.
Y119CIA.l knock-down produces strong additive effect on embryonic lethality of a
mutant of mom-4, a gene upstream of lit-I: The identification of penetrant genetic
modifiers that are specific, and affect multiple different alleles of lit-l suggests that both
modifiers influence lit-l dependent processes, but does not provide information about
where or what the genes are directly influencing. The gene mom-4 encodes a MAPKKK
protein that has been shown to function upstream of LIT-1 (SHIN et at. 1999). If
Yl19ClA.1 functions in the same or related genetic pathway influencing cell polarity
and cell fate specification, we would expect a significant additive effect on embryonic
lethality when both loci are compromised. We used a ts mutant allele of the gene mom-4
to test for this additive effect. With empty vector RNAi at 20°C, 8% of mom-4(ne1539ts)
embryos fail to hatch. At this temperature, depletion ofYl19ClA.l with RNAi increases
lethality to 56.6% (Figure 3). This result is consistent with Yl19ClA.l having a function
that contributes to tit-l dependent processes.
drh-3 Knock-Down Enhances Embryonic Lethality In mom-4 Mutant: The penetrant
and highly specific suppression of embryonic lethality in three lit-l mutants after RNAi
depletion of drh-3 suggests some type of functional negative regulation on MAPK or
63
100
90
eo 80
t:::
:.§ 70
1d 60
:::r:
E 50Q)
~ 40A...
30
20
10
o
Y119ClA.l RNAi
lit-1 (or131) dhc-1 (or195) spn-4(or191) spd-5(or213)mel-26(or543) rfl-1 (or198)
_ Empty Vector RNAi
Y119C1A.l RNAi
_ Empty Vector RNAi
drh-3 RNAi
drh-3 RNAi100
90
00 80
t:::
:.§ 70
'cd
:::r: 60
~ 50e
~ 40
30
20
10
Ol....liiiliiil.L---.l..J..--;;;=.L-----l~-..=-...!....II__--L
lit-1(or131) dhc-1(or195) spn-4(or191) spd-5(or213)mel-26(or543) rfl-1(or198)
Figure 2.- Specificity analysis of genetic interactions caused by Yll9C lA.l
(enhancer of lit-i) or drh-3 (suppressor of lit-i) RNAi. Data points used to
generate the graphs are averaged data from at least four replicate experiments.
Error bars equal one standard deviation.
64
A
Yl19CIA.l RNAi
100
90
80~
~ 70
::c 60
5 50~
A.. 40
30
20
10
o
B
lit-1 (or13lts) lit-1 (ne1991 ts) lit-1 (or393ts) mom-4(ne1539ts)
_ Empty Vector RNAi
Y119CIA.l RNAi
drh-3 RNAi100
90
OIl 80
!=i
~ 70
~
::c 60
5~ 50
Q)
A.. 40
30
20
10
OL...-_Ll...----l...L..---.l--LL
lit-1(or131ts) lit-1(ne199lts) lit-1(or393ts) mom-4(ne1539ts)
_ Empty Vector RNAi
drh-3 RNAi
Figure 3.- RNAi knockdown ofY119ClA.l (A) and drh-3 (B) effect on embryonic
viability in three different ts alleles of lit-l and one ts allele of mom-4. N>200
embryos for all data points shown.
65
Wnt signaling. To test this further we asked whether RNAi depletion of drh-3 also
suppresses embryonic lethality in mom-4(nei539ts) mutants. We found that depleting
drh-3 gene product with RNAi increases lethality in this mutant background (Figure 3)
suggesting a more complex relationship with LIT-l/MOM-4 signaling (see discussion).
Since drh-3 is itself required for embryonic viability, enhancement of mom-4(nei539ts)
could indicate that it may be functioning to negatively regulate Wnt or MAPK signaling
downstream of mom-4 but upstream of lit-i. Additional genetic and molecular analysis
will be needed to determine the epistatic relationship between these genes.
Y119CIA.l enhances loss of gut and drh-3 suppresses loss of gut: lit-iloss-of-
function mutants fail to produce intestine during embryogenesis. IfYI19CIA.I
specifically influences endoderm specificaton induced by the WntiMAPK pathways, then
depletion ofYI19CIA.I should enhance the loss of intestine phenotype. Indeed we found
a striking enhancement of this phenotype indicating that Y119C1A.I does influence LIT-
1 in a way that affects endoderm specification, presumably through the regulation of
POP-I activity or localization (Figure 4 and Table 1). We also tested if depletion of drh-3
in lit-i(or13lts) mutants suppresses the loss of intestine seen at a highly restrictive
temperature. We observed significant suppression of the loss-of-intestine phenotype with
the depletion of drh-3 (Figure 4 and Table 1).
lit-l(orI31) at
23°C
lit-l (or131)
Y1l9CIA.l
RNAi at 23°C
lit-l (or131) at
25°C
lit-l (or131)
drh-3 RNAi at
25°C
DIC
Dark Field
Polarized Light
66
Figure 4.- Gut granules serve as a marker for endodermal development
TABLE 1
Percent embryos with gut granules
Control RNAi Y119CIA.l RNAi
lit-l (or131 ts)
at 23°C 49% N=269 8% N=369
Control RNAi drh-3 RNAi
lit-l (or131 ts)
at 25°C 9.5% N=429 51% N=305
67
GFP::Y119CIA.llocalizes to nuclear membranes in the early embryo: To gain
further insight into how Y119CIAl may influence WNTIMAPK signaling we
constructed an N-terminal GFP fusion construct ofYI19CIAI. We isolated transformed
GPP expressing lines and analyzed the localization patterns during early cell divisions.
We observed cytoplasmic and nuclear localization ofGFP::YI19CIA.l in all early cells.
During mitosis and early interphase we observed diffuse cytoplasmic localization. During
late interphase and nuclear envelope breakdown, the fusion protein localized to the nuclei
of all cells. Initially, signal is seen surrounding the nuclei similar to nuclear pore proteins,
but then becomes more nuclear shortly before NEB (Figure 5). Localization to the
nueleus is supportive of Yl19C IAI somehow functioning to regulate nuclear-
cytoplasmic transport of Wnt and/or MAPK components.
DISCUSSION
We have used feeding RNAi to conduct a large-scale screen for enhancers and
suppressors of lit-1 (or131ts) embryonic lethality. Quantitative analysis eliminated all but
two genes that modify embryonic lethality in lit-1 (or131ts) mutants, but not in other
unrelated ts embryonic-lethal mutants. Here we report the identification of two genes that
both have a strong and specific interaction with mutants of lit-1 and mom-4, and appear to
playa role in endoderm specification in C. elegans.
Figure 5.- Expression ofGFP fusion to Y119CIA.l encoded protein in early
embryos from transgenic strain. All images are from the same embryo at
various (unspecified) time-points during early embryonic cell divisions.
Frames from six optical sections were projected from spinning-disk confocal
time-lapse videomicrographs. Anterior is to the left. Long arrow indicates
nuclear signal. White arrowhead indicates nuclear envelope signal. Short
arrow indicates P granules.
68
69
Y119CIA.l: A strong candidate for regulating LIT-l nucleo-cytoplasmic transport:
While Yl19ClAl has no previously known function or knockdown phenotype, we show
that this gene is essential for both viability and endoderm formation in three different ts
lit-l mutant strains grown at semi-permissive temperatures. RNAi mediated knock-down
also strongly enhances mutants of mom-4, a protein known to function upstream of LIT-l
(SHIN et al. 1999). The gene Yl19C lAl encodes a novel protein containing a RanBP-
Type 2 Zinc Finger domain near the C-terminus. This domain is found in five different
worm genes including the nuclear pore proteins npp-7 and npp-9/NUP358/RanBP2.
Mammalian RanBP2/Nup358 is a protein that is comprised largely of a repetitive series
ofRanBP2 zinc finger domains, and is one of three known nuclear pore components that
reside on the cytoplasmic face of the nuclear pore complex (DELPHIN et al. 1997).
Nup358 is thought to be the major, if not the only, component of the cytoplasmic
filaments that extend from the nuclear pore out into the cytoplasm. The zinc finger
domains ofNup358 have been shown to associate with the nuclear export factor CRMI
and RNAi mediated knockdown ofNup358 in mouse blastocysts cause a loss ofCRM-l
localization at the cytoplasmic side of the nuclear pore (BERNAD et al. 2004).
Furthermore, RNAi mediated knock-down ofNup358 in MCF-7 cells causes a partial,
but significant reduction in nuclear export ofa reporter construct (BERNAD et al. 2004).
Importantly, the authors state that in experiments in which RNAi was less efficient
«90% knock-down ofprotein expression), little or no loss of nuclear export was
observed. This finding indicates that only a small of amount ofNup358 is sufficient for
nuclear export. If Yll9C1Al has a similar role in mediating export of nuclear factors,
70
the lack of lethality by RNAi in a wildtype background may be simply because of
insufficient knock-down of the gene product. Likewise it would make sense that a mutant
of a nuclear export substrate (potentially LIT-1) would produce an additive negative
effect and result in lethality.
Additional support for YII9CIA.I acting to regulate nuclear localization ofLIT-
1, comes from a large scale Y2H screen of metazoan specific C. eZegans genes (LI et aZ.
2004), which showed YII9CIA.l is a reciprocal binding partner ofLIT-I. This apparent
physical interaction combined with the very pentrant and specific enhancement of both
lit-l and mom-4 mutants strongly suggests that YII9C IA.I has a direct role in
Wnt/MAPK signaling, and likely has some direct interaction with LIT-I. Importantly, the
Y 1I9C1A.I bait construct also interacted with nine additional proteins, six of which are
nuclear associated proteins. Five of these nuclear associated proteins (RPY-I, NHR-III,
EYA-I, TAF-9, VAB-3) are transcription factors while the other is a component of the
nuclear transport machinery: IMA-3 (lmportin Alpha nuclear transport factor). Finally,
we showed that GFP::YII9CIA.Ilocalizes to the nuclear envelope consistent with it
functioning at the nuclear pore complex. Additional experiments will be required to
confirm this hypothesis.
Potential role for siRNA mediated regulation ofwnt signaling: We show that
depletion of drh-3, suppresses lethality in lit-l mutants, yet curiously enhances lethality
in a mutant of mom-4. While we cannot yet predict a specific role for this essential and
conserved gene, our data suggests a direct and novel function for this gene in WntiMAPK
71
signaling and/or endoderm specification in C. elegans. drh-3 encodes a Dicer-related
helicase that interacts with Dicer, is required for RNAi in the germline, and for the
production of endogenous small interfereing RNAs (siRNA) (DUCHAINE et al. 2006).
drh-3 is essential for embryonic viability as both RNAi and deletion mutants are lethal
(RNAi produces partially penetrant lethality). Phenotype analysis of drh-3 mutants show
multinucleate germ cells often resulting in an overall sterile phenotype, as well as lagging
mitotic chromosome and anaphase bridges in mitosis (DUCHAINE et al. 2006). A recent
report showed that drh-3 may be required for an siRNA pathway that regulates Ras
signaling in developing larvae. It is possible that some small RNA species generated by a
DRH-3 RNAi complex could regulate the expression ofWnt or MAPK signaling
components. Additional genetic epistasis analysis, localization analysis and identification
of additional DRH-3 generated siRNAs will be needed to pursue this possibility. It would
be interesting to evaluate protein levels of several Wnt or MAPK signaling components
in a background deficient ofDRH-3. IdentifYing a significant change in protein levels of
any of these components (most likely an increase), could indicate siRNA mediated
regulation of their expression. Alternatively, DRH-3 may have an as yet undiscovered
function, independent of its siRNA generating activity.
Concluding remarks: Using a mutant of the MAPK lit-l we have identified two genes
that function to modifY lit-l mutant induced embryonic lethality in a way that also affects
endoderm specification. The enhancement of lethality caused by depleting Y119C1A.1 in
lit-l mutants, but the absence of any phenotype in a wildtype background, is a striking
72
example of the power of modifier screening in the identifying gene function. We do not
yet have defined roles for either drh-3 or Yl19CIA.l, but the evidence presented here
clearly shows that both genes have functions that influence Wnt signaling in C. elegans
embryos.
73
CHAPTER V
CONCLUSIONS
The completion of genome sequencing projects has presented researchers with the
comprehensive genetic code of many different organisms including that of humans.
These projects represent an incredible achievement in the pursuit of understanding the
heritable factors required for life. An even greater challenge will be to identify the
functions for each gene contained in the vast sequences of DNA we now have access to.
This includes identifying how genes interact with each other to coordinate complex
processes that require the proper function and interplay of many proteins. Model
organisms such as C. elegans provide us with a valuable resource to study the function of
genes and genetic interactions, that can then be used to gain insight into the biology and
disease mechanisms of more complex organisms including humans.
Conditional mutant modifier screening: The goal of my dissertation was to identify
novel genetic requirements for essential processes that control embryogenesis in C.
elegans. To do this I used temperature-sensitive mutants defective in specific processes
that occur in the early embryo and screened them with a genomic RNAi library for genes
that, when depleted with RNAi, modify the amount of lethality caused by the mutation.
We identified ~70 reproducible genetic interactions from screening three ts mutants. For
modifiers of the Wnt signaling defective mutant lit-1 (or131ts) , and the neddylation
74
defective mutant rfl-l (or198ts), we showed that quantitatively assessing the specificity of
genetic interactions is crucial step in beginning the processes of characterizing the nature
of genetic interactions. After identifying the most specific and penetrant modifiers we
proceeded to characterize the modifier loci using various techniques.
We show that specific suppressors of rfl-l mutants encode proteins that have
localization patterns similar to many of the components of the neddylation pathway. We
also show that knocking down expression of the strongest suppressors of rfl-l (or198ts)
significantly reduces the cellular defects caused by the mutation. We identified two
highly penetrant enhancers of lethality in rfl-l mutants that produce little or no lethality
in a wildtype background or in ts mutants of unrelated genes. These enhancers failed to
increase early embryonic defects associated with rfl-l mutants. Further analysis showed
the enhancement was likely due to an as yet undiscovered function for rfl-l later in
development that was only detectable when both rfl-l, and the enhancer gene were
reduced in function. In sum, we identified novel roles for 17 conserved genes, validating
the usefulness and efficiency of ts mutant modifier screening.
In chapter IV we presented data describing the interaction between mutants of tit-
1 and depletion of drh-3 and Yl19ClA.l gene products. We show that lit-l ts mutant
lethality, at largely permissive temperature, is dramatically enhanced by RNAi mediated
depletion ofYl19ClA.1. This enhancement is completely specific for mutants of lit-l
and the related gene mom-4, as they fail to enhance any of the five additional ts
embryonic lethal mutants of unrelated genes that we tested. Sequence analysis indicated
the presence of a conserved zinc finger motif, found in specific proteins that function in
75
the nuclear pore complex (NPC). While Y119CIAI is uncharacterized, it was identified
as a reciprocal binding partner of LIT-1 in a large-scale 2-hybrid screen. Additional
targets identified in this 2-hybrid screen are nuclear factors including one component of
the nuclear transport machinery, supporting a possible role in regulating nucleo-
cytoplasmic transport. We show a localization pattern consistent with this possibility as
GFP::YI19CIA.llocalizes to the nuclear membrane. Finally we show a striking
enhancement of the loss-of-intestine phenotype characteristic of lit-i mutants and other
Wnt signaling components that act upstream ofpop-i. Together these findings show that
Y119CIAI has an important role in WntiMAPK signaling. This role was completely
illusive in previous RNAi studies and may indicate that RNAi mediated knockdown of
Y119C IAl is insufficient for causing a phenotype, or that Y119C1Al is simply not
essential for proper development in an otherwise wildtype background. Both of these
scenarios support the importance of modifier screening in determining gene functions.
The gene drh-3 has been previously characterized as essential for development
and is required for RNAi in the germline (DUCHAINE et at. 2006). We show here that
knockdown of drh-3 causes strong and specific suppression of lit-i mutant induced
embryonic lethality. The suppression of lethality also correlates with suppression of the
loss-of-intestine phenotype characteristic of lit-i mutants. Recent functional
characterization of drh-3 indicates important roles in the production of siRNAs and
siRNA mediated regulation of gene expression. The identification of drh-3 as a
suppressor of lit-i mutants shows that the screening method used here can also detect
new roles for essential genes. While we cannot easily predict any specific mechanisms
that explain the suppression of lit-l mutants, a role for siRNA pathways in regulating
Wnt signaling would be a major discovery in the study of these important molecular
pathways.
76
APPENDIX
Summary ofrjl-I(orI98ts) modifiers
77
Gene Encoded Interaction Essentia Specifi Human Blast P %
Protein 1 c Homolo value lengt
g h
csn-5 COP9 suppressor yes yes COPS5 5.2e- 84.8
Signalosome 100
subunit
csn-2 COP9 suppressor yes yes COPS2 6.81e- 83.8
Sinalosome 151
subunit
mus-IOI Nucleotide suppressor yes yes TOPBP 3e-50 61.7
Excision 1
Repair Factor
R10D12. Unknown suppressor yes yes TNRC1 6.4e-06 25.7
14 Function 5
mnk-I Kinase suppressor no yes Q9BUB 2.1e-31
5-2
F25H5.5 Claspin suppressor yes yes CLSPN 1.4e-25 91.3
(kinase)
F22B8.7 Fe-S Protein suppressor no yes MOSC 8.6e-43 85.5
1
T24C4.3 Novel suppressor no yes
cdr-2 glutathione S- suppressor no yes C60rfl 7.3e-22 82.7
transferase- 68
like
T26E3.6 Fibrillin suppressor no yes SORL1 0.0001 84.5
related protein
elks-I Rab6 GTPase- suppressor no yes ERC2 6.4e-36 77.3
interacting
protein
sri-63 7TM suppressor no yes CCR1 0.011 87.7
chemoreceptor
gcy-20 guanylate suppressor no yes NPR1 1.4e- 89
cyclase 133
Igc-20 Acetylcholine suppressor no yes CHRN 1.8e-07 71.6
receptor B1
ugt-I6 UDP- suppressor no yes UGT2B 1.6e-43 95.5
glucuronosyl 7
transferase
78
col-I 64 Collagen suppressor no yes Q59HB 6.4e-43 73
protein 5
vig-I RNA Binding suppressor yes no Q8NC5 2.1e-31 97.9
Protein 1-4
M106.4 GMP suppressor no no GMPS 2.7e- 74.7
Synthase 151
aqp-5 Aquaporin suppressor no no AQP8 6.4e-26 73.8
T05H4.11 Novel suppressor no no
T04C9.1 Rho GTPase- suppressor no no ARHG 3.1e- 79
activating AP10 144
protein
sel-IV F-Box,WD40 enhancer no yes FBXW 1.ge- 76.7
Protein 7 126
pha-I novel enhancer yes/no yes Q96Q8 0.018 71.1
9-3
R05A10. novel enhancer no yes
8
srh-292 7TM enhancer no yes LMBR .071 75.8
chemoreceptor 1L
T14E8.1 Protein enhancer no yes MET 1.8e-48 50.8
tyrosine
kinase
C24D10. Protein enhancer no yes Q12923 1.2e-06 50.6
1 tyrosine -2
phosphatase
abcf-3 ABC enhancer no no ABCF3 1.3e- 98
transporter 183
F32B6.9 RNA splicing enhancer yes/no no PRPF1 6e-58 96.6
factor 8
gpd-4 glyceraldehyd enhancer yes no GAPD 3.2e- 99.4
e-3-phosphate H 132
dehydrogenas
es
cutl-8 Cuticulin enhancer no no MAST1 .066 50.9
precursor
ZK686.4 Zinc finger enhancer no no ZMAT 3.4e-50 96.8
protein 2
mtm-3 Myotubularin- enhancer no no Q13615 1.5e-74 64.1
related protein -2
T01C3.2 Chromatin enhancer no no MIT:E 0.0014 92.9
modifying NY2-
protein 001
79
REFERENCES
ALBERTSON, D. G., and J. N. THOMSON, 1993 Segregation ofholocentric chromosomes at
meiosis in the nematode, Caenorhabditis elegans. Chromosome Res. 1: 15-26.
ANGERS, S., C. J. THORPE, T. L. BIECHELE, S. J. GOLDENBERG, N. ZHENG et aI., 2006
The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin
pathway by targeting Dishevelled for degradation. Nat. Cell BioI. 8: 348-357.
BAUGH, L. R., J. C. WEN, A. A. HILL, D. K. SLONIM, E. L. BROWN et aI., 2005 Synthetic
lethal analysis of Caenorhabditis elegans posterior embryonic patterning genes
identifies conserved genetic interactions. Genome BioI. 6: R45.
BERNAD, R., H. VAN DER VELDE, M. FORNEROD and H. PICKERSGILL, 2004
Nup358/RanBP2 attaches to the nuclear pore complex via association with Nup88
and Nup214/CAN and plays a supporting role in CRM1-mediated nuclear protein
export. Mol. Cell BioI. 24: 2373-2384.
BORNSTEIN, G., D. GANOTH and A. HERSHKO, 2006 Regulation ofneddylation and
deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F-box protein and
substrate. Proc. Natl. Acad. Sci. USA 103: 11515-11520.
BOWERMAN, B., and T. KURZ, 2006 Degrade to create: developmental requirements for
ubiquitin-mediated proteolysis during early C. elegans embryogenesis.
Development 133: 773-784.
BRENNER, S., 1974 The genetics ofCaenorhabditis elegans. Genetics 77: 71-94.
CADIGAN, K. M., and R. NUSSE, 1997 Wnt signaling: a common theme in animal
development. Genes Dev. 11: 3286-3305.
CULLINAN, S. B., 1. D. GORDAN, J. JIN, J. W. HARPER and J. A. DIEHL, 2004 The Keap1-
BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase: oxidative
stress sensing by a Cu13-Keap1ligase. Mol. Cell. BioI. 24: 8477-8486.
DELPHIN, C., T. GUAN, F. MELCHIOR and L. GERACE, 1997 RanGTP targets p97 to
RanBP2, a filamentous protein localized at the cytoplasmic periphery of the
nuclear pore complex. Mol. BioI. Cell 8: 2379-2390.
DETWILER, M. R., M. REUBEN, X. LI, E. ROGERS and R. LIN, 2001 Two zinc finger
proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in
C. elegans. Dev. CellI: 187-199.
80
Dow, M. R., and P. E. MAINS, 1998 Genetic and molecular characterization of the
Caenorhabditis elegans gene, mel-26, a postmeiotic negative regulator of mei-l, a
meiotic-specific spindle component. Genetics 150: 119-128.
DUCHAINE, T. F., J. A. WOHLSCHLEGEL, S. KENNEDY, Y. BEl, D. CONTE, JR. et aI., 2006
Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in
multiple small-RNA-mediated pathways. Cell 124: 343-354.
FAY, D. S., X. QIU, E. LARGE, C. P. SMITH, S. MANGO et al., 2004 The coordinate
regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-l, and ubc-
18. Dev. BioI. 271: 11-25.
FEINBERG, E. H., and C. P. HUNTER, 2003 Transport of dsRNA into cells by the
transmembrane protein SID-I. Science 301: 1545-1547.
FIRE, A., S. Xu, M. K. MONTGOMERY, S. A. KOSTAS, S. E. DRIVER et aI., 1998 Potent
and specific genetic interference by double-stranded RJ~A in Caenorhabditis
elegans. Nature 391: 806-811.
FRASER, A., 2004 Towards full employment: using RNAi to find roles for the redundant.
Oncogene 23: 8346-8352.
GEYER, R., S. WEE, S. ANDERSON, J. YATES and D. A. WOLF, 2003 BTB/POZ domain
proteins are putative substrate adaptors for cullin 3 ubiquitin ligases. Mol. Cell
12: 783-790.
GOMES, J. E., S. E. ENCALADA, K. A. SWAN, C. A. SHELTON, J. C. CARTER et al., 2001
The maternal gene spn-4 encodes a predicted RRM protein required for mitotic
spindle orientation and cell fate patterning in early C. elegans embryos.
Development 128: 4301-4314.
GUlL, S., and M. ESTELLER, 2008 DNA methylomes, histone codes and miRNAs: Tying
it all together. lnt. J. Biochem. Cell BioI.
Guo, S., and K. J. KEMPHUES, 1995 par-l, a gene required for establishing polarity in C.
elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically
distributed. Cell 81: 611-620.
HAMILL, D. R., A. F. SEVERSON, J. C. CARTER and B. BOWERMAN, 2002 Centrosome
maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein
with multiple coiled-coil domains. Dev. Cell 3: 673-684.
81
HETFELD, B. K., A. PETH, X. M. SUN, P. HENKLEIN, G. M. COHEN et aI., 2008 The COP9
signa10some-mediated deneddylation is stimulated by caspases during apoptosis.
Apoptosis 13: 187-195.
HOLWAY, A. H., C. HUNG and W. M. MICHAEL, 2005 Systematic, RNA-interference-
mediated identification of mus-l 01 modifier genes in Caenorhabditis elegans.
Genetics 169: 1451-1460.
HUBBARD, E. J., G. Wu, J. KITAJEWSKI and 1. GREENWALD, 1997 sel-l0, a negative
regulator of lin-12 activity in Caenorhabditis elegans, encodes a member of the
CDC4 family ofproteins. Genes Dev. 11: 3182-3193.
HUNT-NEWBURY, R., R. VIVEIROS, R. JOHNSEN, A. MAH, D. ANASTAS et al., 2007 High-
throughput in vivo analysis of gene expression in Caenorhabditis elegans. PLoS
BioI. 5: e237.
JADHAV, S., M. RANA and K. SUBRAMANIAM, 2008 Multiple maternal proteins
coordinate to restrict the translation of C. elegans nanos-2 to primordial germ
cells. Development 135: 1803-1812.
JAGER, S., H. T. SCHWARTZ, H. R. HORVITZ and B. CONRADT, 2004 The Caenorhabditis
elegans F-box protein SEL-1 0 promotes female development and may target
FEM-1 and FEM-3 for degradation by the proteasome. Proc. NatI. Acad. Sci. U S
A 101: 12549-12554.
JONES, D., and E. P. CANDIDO, 2000 The NED-8 conjugating system in Caenorhabditis
elegans is required for embryogenesis and terminal differentiation of the
hypodermis. Dev. BioI. 226: 152-165.
JOUKOV, V., A. C. GROEN, T. PROKHOROVA, R. GERSON, E. WHITE et aI., 2006 The
BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly.
Cell 127: 539-552.
KAMATH, R. S., and J. AHRINGER, 2003 Genome-wide RNAi screening in
Caenorhabditis elegans. Methods 30: 313-321.
KELLY, W. G., and A. FIRE, 1998 Chromatin silencing and the maintenance ofa
functional germline in Caenorhabditis elegans. Development 125: 2451-2456.
KIDD, A. R., 3RD, 1. A. MISKOWSKI, K. R. SIEGFRIED, H. SAWA and J. KIMBLE, 2005 A
beta-catenin identified by functional rather than sequence criteria and its role in
WntiMAPK signaling. Cell 121: 761-772.
82
KURZ, T., N. OZLU, F. RUDOLF, S. M. O'ROURKE, B. LUKE et ai., 2005 The conserved
protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S.
cerevisiae. Nature 435: 1257-1261.
KURZ, T., L. PINTARD, J. H. WILLIS, D. R. HAMILL, P. GONCZY et al., 2002 Cytoskeletal
regulation by the Nedd8 ubiquitin-like protein modification pathway. Science
295: 1294-1298.
LABBE, J. C., and B. GOLDSTEIN, 2002 Embryonic development: a new SPN on cell fate
specification. Curro BioI. 12: R396-398.
LABBE,1. c., A. PACQUELET, T. MARTY and M. GOTTA, 2006 A genomewide screen for
suppressors ofpar-2 uncovers potential regulators ofPAR protein-dependent cell
polarity in Caenorhabditis elegans. Genetics 174: 285-295.
LAMITINA, T., C. G. HUANG and K. STRANGE, 2006 Genome-wide RNAi screening
identifies protein damage as a regulator of osmoprotective gene expression. Proc.
NatI. Acad. Sci. USA 103: 12173-12178.
LANDER, E. S., L. M. LINTON, B. BIRREN, C. NUSBAUM, M. C. ZODY et ai., 2001 Initial
sequencing and analysis of the human genome. Nature 409: 860-921.
LEHNER, B., C. CROMBIE, J. TISCHLER, A. FORTUNATO and A. G. FRASER, 2006
Systematic mapping of genetic interactions in Caenorhabditis elegans identifies
common modifiers of diverse signaling pathways. Nat. Genet. 38: 896-903.
LI, S., C. M. ARMSTRONG, N. BERTIN, H. GE, S. MILSTEIN et al., 2004 A map of the
interactome network of the metazoan C. elegans. Science 303: 540-543.
Lo, M. C., F. GAY, R. ODOM, Y. SHI and R. LIN, 2004 Phosphorylation by the beta-
cateniniMAPK complex promotes 14-3-3-mediated nuclear export ofTCFIPOP-1
in signal-responsive cells in C. elegans. Cell 117: 95-106.
LOPEZ-GIRONA, A., B. FURNARI, O. MONDESERT and P. RUSSELL, 1999 Nuclear
localization ofCdc25 is regulated by DNA damage and a 14-3-3 protein. Nature
397: 172-175.
Lu, C., and P. E. MAINS, 2007 The C. elegans anaphase promoting complex and MBK-
2/DYRK kinase act redundantly with CUL-3/MEL-26 ubiquitin ligase to degrade
MEI-1 microtubule-severing activity after meiosis. Dev. BioI. 302: 438-447.
LYAPINA, S., G. COPE, A. SHEVCHENKO, G. SERINO, T. TSUGE et ai., 2001 Promotion of
NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science 292: 1382-
1385.
83
MADURO, M. F., R. LIN and J. H. ROTHMAN, 2002 Dynamics ofa developmental switch:
recursive intracellular and intranuclear redistribution of Caenorhabditis elegans
POP-1 parallels Wnt-inhibited transcriptional repression. Dev. BioI. 248: 128-
142.
MAEDA, 1., Y. KOHARA, M. YAMAMOTO and A. SUGIMOTO, 2001 Large-scale analysis of
gene function in Caenorhabditis elegans by high-throughput RNAi. Curf. BioI.
11: 171-176.
MAINS, P. E., K. 1. KEMPHUES, S. A. SPRUNGER, 1. A. SULSTON and W. B. WOOD, 1990
Mutations affecting the meiotic and mitotic divisions of the early Caenorhabditis
elegans embryo. Genetics 126: 593-605.
MANKE, 1. A., D. M. LOWERY, A. NGUYEN and M. B. YAFFE, 2003 BRCT repeats as
phosphopeptide-binding modules involved in protein targeting. Science 302: 636-
639.
McNALLY, K., A. AUDHYA, K. OEGEMA and F. 1. McNALLY, 2006 Katanin controls
mitotic and meiotic spindle length. J. Cell BioI. 175: 881-891.
MEINNEL, T., and C. GIGLIONE, 2008 Protein lipidation meets proteomics. Front. Biosci.
13: 6326-6340.
MENEGHINI, M. D., T. ISHITANI, J. C. CARTER, N. HISAMOTO, J. NINOMIYA-TsUJI et ai.,
1999 MAP kinase and Wnt pathways converge to downregulate an HMG-domain
repressor in Caenorhabditis elegans. Nature 399: 793-797.
NANDI, D., P. TAHILIANI, A. KUMAR and D. CHANDU, 2006 The ubiquitin-proteasome
system. J. Biosci. 31: 137-155.
O'ROURKE, S. M., M. D. DORFMAN, J. C. CARTER and B. BOWERMAN, 2007 Dynein
modifiers in C. elegans: light chains suppress conditional heavy chain mutants.
PLoS Genet. 3: e128.
Om, S. L., X. PAN, B. D. PEYSER, P. YE, P. B. MELUH et ai., 2006 Global synthetic-
lethality analysis and yeast functional profiling. Trends Genet. 22: 56-63.
PIANO, F., A. 1. SCHETTER, D. G. MORTON, K. C. GUNSALUS, V. REINKE et al., 2002
Gene clustering based on RNAi phenotypes of ovary-emiched genes in C.
elegans. Curf. BioI. 12: 1959-1964.
84
PINTARD, L., T. KURZ, S. GLASER, J. H. WILLIS, M. PETER et ai., 2003a Neddylation and
deneddylation ofCUL-3 is required to target MEI-l/Katanin for degradation at
the meiosis-to-mitosis transition in C. eiegans. Curf. BioI. 13: 911-921.
PINTARD, L., J. H. WILLIS, A. WILLEMS, J. L. JOHNSON, M. SRAYKO et ai., 2003b The
BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-
ligase. Nature 425: 311-316.
PRAITIS, V., 2006 Creation of transgenic lines using microparticle bombardment
methods. Methods Mol. BioI. 351: 93-107.
PRIESS, J. R, H. SCHNABEL and R. SCHNABEL, 1987 The gip-llocus and cellular
interactions in early C. eiegans embryos. Cell 51: 601-611.
QIU, x., and D. S. FAY, 2006 ARI-l, an RBR family ubiquitin-ligase, functions with
UBC-18 to regulate pharyngeal development in C. eiegans. Dev. BioI. 291: 239-
252.
QUINTIN, S., P. E. MAINS, A. ZINKE and A. A. HYMAN, 2003 The mbk-2 kinase is
required for inactivation of IvlEI-l/katanin in the one-cell Caenorhabditis eiegans
embryo. EMBO Rep. 4: 1175-1181.
ROCHELEAU, C. E., W. D. DOWNS, R LIN, C. WITTMANN, Y. BEl et ai., 1997 Wnt
signaling and an APC-related gene specify endoderm in early C. eiegans
embryos. Cell 90: 707-716.
ROCHELEAU, C. E., J. YASUDA, T. H. SHIN, R LIN, H. SAWA et ai., 1999 WRM-l
activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals
in C. eiegans. Cell 97: 717-726.
SAROV, M., S. SCHNEIDER, A. POZNIAKOVSKI, A. ROGUEV, S. ERNST et ai., 2006 A
recombineering pipeline for functional genomics applied to Caenorhabditis
eiegans. Nat. Methods 3: 839-844.
SCHELLENBERG, M. J., D. B. RITCHIE and A. M. MACMILLAN, 2008 Pre-mRNA splicing:
a complex picture in higher definition. Trends Biochem. Sci. 33: 243-246.
SCHWECHHEIMER, C., G. SERINO, J. CALLIS, W. L. CROSBY, S. LYAPINA et ai., 2001
Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in
mediating auxin response. Science 292: 1379-1382.
SHANER, N. C., R. E. CAMPBELL, P. A. STEINBACH, B. N. GIEPMANS, A. E. PALMER et
ai., 2004 Improved monomeric red, orange and yellow fluorescent proteins
85
derived from Discosoma sp. red fluorescent protein. Nat. BiotechnoI. 22: 1567-
1572.
SHIN, T. H., J. YASUDA, C. E. ROCHELEAU, R. LIN, M. SOTO et al., 1999 MOM-4, a MAP
kinase kinase kinase-related protein, activates WRM-l/LIT-1 kinase to transduce
anterior/posterior polarity signals in C. elegans. Mol. Cell 4: 275-280.
SIMMER, F., C. MOORMAN, A. M. VANDERLINDEN, E. KUIJK, P. V. VANDEN BERGHE et
al., 2003 Genome-wide RNAi of C. elegans using the hypersensitive rrf3 strain
reveals novel gene functions. PLoS BioI. 1: E12.
SONNICHSEN, B., L. B. KOSKI, A. WALSH, P. MARSCHALL, B. NEUMANN et al., 2005
Full-genome RJ'JAi profiling of early embryogenesis in Caenorhabditis elegans.
Nature 434: 462-469.
SRAYKO, M., D. W. BUSTER, O. A. BAZIRGAN, F. J. McNALLY and P. E. MAINS, 2000
MEI-1/MEI-2 katanin-like microtubule severing activity is required for
Caenorhabditis elegans meiosis. Genes Dev. 14: 1072-1084.
STITZEL, M. L., K. C. CHENG and G. SEYDOUX, 2007 Regulation ofMBK-2/Dyrk kinase
by dynamic cortical anchoring during the oocyte-to-zygote transition. CUff. BioI.
17: 1545-1554.
THORPE, C. J., A. SCHLESINGER, J. C. CARTER and B. BOWERMAN, 1997 Wnt signaling
polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm.
Cell 90: 695-705.
TIMMONS, L., D. L. COURT and A. FIRE, 2001 Ingestion of bacterially expressed dsRNAs
can produce specific and potent genetic interference in Caenorhabditis elegans.
Gene 263: 103-112.
TIMMONS, L., and A. FIRE, 1998 Specific interference by ingested dsRNA. Nature 395:
854.
TONG, A. H., G. LESAGE, G. D. BADER, H. DING, H. Xu et al., 2004 Global mapping of
the yeast genetic interaction network. Science 303: 808-813.
VAN HAAFTEN, G., R. ROMEIJN, J. POTHOF, W. KOOLE, L. H. MULLENDERS et a!., 2006
Identification of conserved pathways of DNA-damage response and radiation
protection by genome-wide RNAi. CUff.BioI. 16: 1344-1350.
WARMING, S., N. COSTANTINO, D. L. COURT, N. A. JENKINS and N. G. COPELAND, 2005
Simple and highly efficient BAC recombineering using galK selection. Nucleic
Acids Res. 33: e36.
86
WU, G., S. LYAPINA, 1. DAS, J. LI, M. GURNEY et al., 2001 SEL-10 is an inhibitor of
notch signaling that targets notch for ubiquitin-mediated protein degradation.
Mol. Cell BioI. 21: 7403-7415.
XING, Y, W. K. CLEMENTS, D. KIMELMAN and W. Xu, 2003 Crystal structure of a beta-
cateninlaxin complex suggests a mechanism for the beta-catenin destruction
complex. Genes Dev. 17: 2753-2764.
Xu, L., Y. WEI, 1. REBOUL, P. VAGLIO, T. H. SHIN et al., 2003 BTB proteins are
substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing
CUL-3. Nature 425: 316-321.
YAMAMOTO, R. R, 1. M. AXTON, Y. YAMAMOTO, R. D. SAUNDERS, D. M. GLOVER etal.,
2000 The Drosophila mus101 gene, which links DNA repair, replication and
condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1
C-terminus domains. Genetics 156: 711-721.
YOST, C., M. TORRES, J. R MILLER, E. HUANG, D. KIMELMAN et al., 1996 The axis-
inducing activity, stability, and subcellular distribution of beta-catenin is
regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev. 10:
1443-1454.
Yu, X., C. C. CHINI, M. HE, G. MER and J. CHEN, 2003 The BRCT domain is a phospho-
protein binding domain. Science 302: 639-642.