Talk on Friday 01:48-02:00pm submitted by Jagjit Singh
Significance of novel RNA-protein interaction in U12-dependent splicing
Jagjit Singh MS (Center of Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH-44115), Kavleen Sikand Ph.D., Tupa Basu Roy M.S, Girish C. Shukla Ph.D.
Recently, a second class of spliceosome that removes a distinct set of introns was revealed. Even though minor class introns constitute only <1 % of the total introns, their host genes are involved in very critical cellular functioning for example cell proliferation, DNA replication and repair. U12 and U6atac snRNAs, the functional analogs of U2 and U6 of major class spliceosome, play a pivotal role in the removal of U12-dependent introns by performing almost similar RNA-RNA interactions with the intron. In addition, U6atac-U12 interaction is known to establish an intermolecular base paired helix I region. Beyond these known functional regions of U6atac and U12 snRNAs, several regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that 3’ stem-loop region of U6atac snRNA serves as U12-dependent spliceosome specific guide element. In this work, we show the functional requirement of an evolutionary redundant substructure of U6atac 3’ stem-loop in minor class in vivo splicing. Our in vitro data show that C-terminal RRM (RNA recognition motif) of p65, U11/U12 18s complex protein, interacts with distal stem-loop substructure of U6atac 3’ stem-loop. Further, we show that the functionally important p65 protein-binding apical stem-loop of U12 snRNA can be replaced by U6atac stem-loop. Using binary splice site mutation suppressor assay, we demonstrate that the exchange of these RNA elements between U12 and U6atac is functional in in vivo splicing. In addition, we found that structure of this distal 3’ stem loop is more important as compared to the sequence for in vivo splicing. We have also shown that 3’ end of U6atac from distant species with different secondary structure and sequence could catalyze both the splicing as well as the interaction with p65 to different extent, indicating its evolutionarily conserved function. These observations support the idea that both the RNA-RNA as well as the RNA-protein interactions occurring in minor class spliceosome are extremely flexible as compared to major class spliceosome.
Keywords: U12-dependent splicing, U6atac snRNA, p65