Abstract 1724 Structural and Functional Characterization of Yeast Splicing Protein Dib1

Pre-messenger RNA splicing involves the excision of non-coding DNA and the ligation of coding DNA to produce mature mRNA. This process is facilitated by the spliceosome, a multi-megadalton ribonucleoprotein complex. In Saccharomyces cerevisiae, Dib1, a 143 amino acid protein, resides at the catalytic core of the spliceosome and departs in the transition from the pre-catalytic spliceosome to the catalytically active spliceosome. Dib1's departure is crucial for spliceosomal activation. While the mechanism of Dib1's departure is uncharacterized, we propose that the flexible carboxy-tail of Dib1 plays a role in Dib1's association with the splicing machinery. To investigate Dib1's C-terminus in relation to the splicing machinery, truncations to Dib1's C-terminus were constructed and assessed by growth, splicing, and structural assays. Wild-type and truncated versions of Dib1 were isolated by two-step column purification, and their stabilities were assessed using circular dichroism spectroscopy under increasing denaturant concentrations. Yeast growth assays and in vitro splicing reactions were performed to observe the effects of truncations on cellular function and splicing. We show that yeast expressing Dib1 with more than 12 residues removed from Dib1's C-terminus exhibit a temperature-sensitive phenotype and defective splicing once heat-shocked. Our findings also show that Dib1 exhibits a minimal loss of stability with 14 residues off the C-terminus, but additional truncations result in much greater instability. Thus, the protein's stability is not severely reduced until 15 residues have been deleted, but we see changes to yeast growth at only 13 residues deleted. To further explore the importance of residue identity for the amino acids that result in growth defects when truncated, we have introduced alanine substitutions and assessed their growth and stability. We speculate that these residues are engaged in necessary interactions in the core of the spliceosome. Overall, we have demonstrated that the stability of Dib1 is linked to the length of its C-terminus, and we speculate that the C-terminus is critical for the stabilization of the spliceosome complex. Funding Source: This work was supported by Trinity University's Chemistry Department and the Welch Foundation (W-1905 and W-0031).