Poster abstracts

Poster number 55 submitted by Jagjit Singh

Swappable RNA Structures in the Minor Spliceosome

Jagjit Singh (Center of Gene Regulation in Health and Disease, Cleveland State Univ), Kavleen Sikand , Girish C. Shukla

Abstract:
U12 and U6atac snRNAs play a key role in the minor class splicing. U12 binds to the branch point, whereas regions, whereas U6atac binds to the 5' splice site after the U11 interactions are destabilized. The 3' stem loop of the U6atac RNA 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 complex, has two RNA binding domains. Previous data show that the c-terminal domain of p65 interacts 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 give rise 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 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 predicting 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 the plasticity of RNA-RNA base-pairing interactions in U12-dependent spliceosome.

Keywords: U12 snRNA, U6atac snRNA, Splicing