Abstract 1723 Investigation of the Dib1 Splicing Protein in the Reduced Spliceosome of C. merolae

Removal of introns through the process of pre-messenger RNA splicing is facilitated by the spliceosome.The spliceosome is a multi-megadalton ribonucleoprotein complex that undergoes a complex process of assembly and activation.One small but essential protein involved in this process is Dib1, a protein associated with the U5 small nuclear ribonucleoprotein (snRNP).It is known that the departure of Dib1 is necessary for the spliceosome to transition to its catalytically active state, but the cause of its departure remains unknown.This project focuses on the study of Dib1 in Cyanidioschyzon merolae (C.merolae), an extremophile red algae with an extremely reduced spliceosome.The spliceosome of C. merolae contains only 43 core splicing proteins, and of these, two homologs of Dib1 have been discovered.Additionally, the secondary structure of the U5 snRNA which Dib1 is associated with is highly conserved between C. merolae and S. cerevisiae.By studying Dib1 in the reduced spliceosome of C. merolae, the critical interactions of Dib1 along with the importance of its structure and departure will be further explored.To investigate the role of Dib1 in C. merolae, protein stability studies have been performed using circular dichroism spectroscopy to compare the two homologs of C. merolae Dib1 to S. cerevisiae Dib1.Both homologs of C. merolae Dib1 were found to be more stable than yeast Dib1.Additionally, the C. merolae Dib1 homologs, which only differ by four amino acids, had a significantly different stability.To investigate the cause of this difference, mutant proteins were constructed to replace one amino acid individually with the amino acid from the other homolog, and these proteins are being investigated as before.Overall, there are notable differences between the function and stability of Dib1 in S. cerevisiae and C. merolae.The stability of the C. merolae homologs proteins will continue to be compared to uncover what interactions are leading to the stability differences.