Characterization of the Small Splicing Protein Dib1, a Component of the U4/U6‐U5 snRNP

Abstract

The spliceosome is a highly dynamic molecular machine composed of small nuclear ribonucleoprotein particles (snRNP). The snRNP facilitates the joining of exons via two transesterification reactions that remove the non‐coding intronic regions. Once the introns are removed, the resulting mature RNA (mRNA) transcript can then be translated into functional protein. The fidelity of this process is crucial, and mistakes in splicing can lead to diseases such as Retinitis pigmentosa. One of the critical steps in splicing is the activation of the U4/U6‐U5 triple snRNP upon joining the spliceosomal complex.­­ In this study, we focus on the small, essential splicing protein Dib1, whose function in splicing remains unknown. Dib1 is 15kDa protein with a thioredoxin‐like fold. Previous data suggests that Dib1 interacts with splicing proteins that bridge the U4/U6 and U5 snRNPs. We characterized the role of Dib1 by rationally designing dib1 mutants and assaying for effects on Saccharomyces cerevisiae growth. We purified these Dib1 mutant proteins and examined their structures by circular dichroism in order to determine the effects these mutants have on the structure of Dib1 protein. In addition, splicing assays were conducted in order to determine the effects the mutant may have on mediating splicing. These assays will help further characterize Dib1 and its role in splicing as well as lend insight into further understanding the splicing machinery.