RRM
2013 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
U12 and U6atac in minor class are functional analogs of U2 and U6 snRNAs of major class spliceosome and play a pivotal role in splicing of minor class introns. U12 snRNA binds to the branch point whereas U6atac snRNA binds to the 5' splice site after U11 interactions are destabilized. The 3' stem loop of the U6atac snRNA is considered as the guide element in minor class splicing. Various inter- and intramolecular RNA-RNA interactions are facilitated by spliceosomal proteins and other splicing factors. p65 RNA binding protein, a component of U11/U12 di-snRNP 18S complex, has two RNA binding domains. The c-terminal domain of p65 is shown to interact with terminal end of U12 stem-loop (SL) III. Our data also show that the terminal end of the U12 SL III is functionally important for in-vivo splicing. Structural and sequence similarity between the distal 3' SL of U6atac and the apical loop of U12 SL III gives rise to the notion that p65 has potential to interact with U6atac. Our data show that the distal SL of 3' U6atac and terminal loop of U12 SL III are functionally swappable, in vivo. In addition, Electrophoretic Mobility Shift Assay (EMSA) data show that p65 c-terminal domain interacts with the distal SL of 3' U6atac SL in a dose dependent manner. 3' distal U6atac SL mutagenesis analysis revealed that point mutation A99C and C100G in the loop enhanced the splicing whereas U98G did not affect the splicing. Abolishing the predicted stem by disrupting the base-pairing in the distal stem caused splicing defect. Restoration of the entire base pairing in the stem by complementary mutations couldn’t restore the WT splicing. In summary, our data show that p65 can interact with 3' SL of U6atac but its role in splicing has not yet been known.
Keywords: U12 and U6atac, minor class spliceosome, p65
