Widom, JuliaCarstairs, Emily2024-08-302024-08-302024https://hdl.handle.net/1794/2989925 pagesRibonucleic acid, or RNA, is a vital biomolecule responsible for many functions in an organism. Messenger RNA (mRNA) is responsible for carrying the genetic instructions from DNA to protein synthesis, which is responsible for much of the cellular activity needed for life. mRNA is originally synthesized as “precursor” mRNA, called pre-mRNA, and undergoes a modification called splicing within the cell nucleus to develop into mature mRNA. Splicing ensures the mRNA contains the correct sequence of nucleotides for proper protein synthesis. RNA sequence informs its structure, which is intrinsically linked to its function and therefore has been the subject of much research. We have developed a method using single-molecule fluorescence resonance energy transfer (smFRET) that allows us to survey the structure of many RNA sequences in a high throughput manner. Using this method, we have varied an important sequence of a truncated yeast pre-mRNA, Ubc4, involved in splicing called the branchpoint sequence (BPS). We show that small changes in the sequence of the BPS of Ubc4 result in a variety of different structural behaviors. However, the overall behavior of the sampled pre-mRNA mimics the behavior exhibited by a wild-type construct, suggesting that secondary structure formation of pre-mRNAs is not influenced by changes to the BPS. This finding, along with further investigations into Ubc4 sequences and protein interactions, will illuminate pre-mRNA structural dynamics in splicing initiation.en-USCC BY-NC-ND 4.0Ribonucleic acidPre-mRNA splicingBiophysicalSingle moleculeMicroscopyThesis/Dissertation0009-0008-0702-4546