Pre-messenger RNA (pre-mRNA) splicing is the process in which introns, noncoding regions of DNA, are removed and exons, coding regions of DNA, are ligated to produce a mature mRNA transcript. This process is mediated by the spliceosome, a dynamic macromolecular complex composed of five small nuclear ribonucleoproteins and other protein factors. At the catalytic center of the splicing machinery is the evolutionarily conserved yeast protein, Dib1, composed of 143 amino acids essential for splicing and cell viability. Dib1 and its human ortholog, hDim1, have demonstrated peptidase activity, specifically autocleavage of the last thirteen and fourteen amino acids, respectively, of their carboxy-terminal tails. The mechanism and function of this autocleavage activity of the carboxy-terminal tail is uncharacterized. Previous yeast growth assays comparing mutants to wildtype Dib1 have established that deletion of fifteen and sixteen amino acids off the carboxy-terminal tail results in temperature-sensitivity of yeast cell growth at 37 masculineC. Since Dib1 and splicing are essential for cell viability, the temperature-sensitive mutants suggest that truncations to the carboxy-terminal tail of Dib1 may result in defective splicing. Thus, the carboxy-terminal tail may play a direct role in splicing or destabilize splicing complexes. To address these two possible hypotheses, we are investigating the effects of the carboxy-terminal truncations on Dib1 stability. Protein purification of wild type and a series of truncated Dib1 mutants were performed. Circular dichroism spectroscopy employing increasing concentrations of guanidinium hydrochloride, a protein denaturant, was used to determine the stability of Dib1 and its truncated mutants. Overall, our findings show that Dib1 stability is directly linked to the length of the tail region and lead us to speculate that the tail is critical for stabilization of the spliceosomal complex.

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Journal Article

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Dunn R, Conde A, Lee G, Meeks G, Pittner N, Maeder C

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