Developing a Simple Titration Cell-Based Splicing Assay
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Muscleblind-like 1 (MBNL1) is a protein that regulates the expression of genes at the step known as pre-mRNA splicing or alternative splicing. Cells use alternative splicing to generate multiple mRNAs (and proteins) from a single gene. MBNL1-mediated splicing is dependent on MBNL1’s ability to bind pre-mRNA targets, which is possible through two critical pairs of tandem zinc finger RNA binding motifs. However, these two distinct RNA binding domains (zinc fingers 1-2 and 3-4) have non-equivalent splicing function in MBNL1 dependent alternative splicing events. The zinc finger pair (1-2) has been shown to contribute significantly higher RNA binding affinity and splicing activity for several RNA substrates compared to the zinc finger pair (3-4). Therefore, a splicing factor with greater splicing activity might be constructed by increasing MBNL1’s affinity through the creation of synthetic MBNL1 containing two copies of zinc finger pair (1-2). To test this hypothesis, an MBNL1 variant replacing the zinc finger pair (3-4) domain with another copy of zinc finger pair (1-2) domain was created – MBNL1 (1-2, 1-2). A simple cell-based splicing assay was developed to compare the splicing activities of WT-MBNL1 and MBNL1 (1-2, 1-2). Preliminary results showed that MBNL1 (1-2, 1-2) had similar splicing activity compared to the wild type MBNL1.