Poster abstracts

Poster number 65 submitted by Kavleen Sikand

U12 snRNA structure-function relationship: Identification of essential RNA motifs required for in vivo U12-dependent splicing

Kavleen Sikand (BGES, Cleveland State University), Girish C. Shukla (BGES, Cleveland State University)

Abstract:
U12 snRNA binds to the branch site sequence of U12-dependent spliceosomal introns by Watson-Crick base pairing and is essential for splicing. Although much is known about U12 snRNA region which base pairs with the branch site of minor class introns, virtually nothing is known about the functional role of other regions of the snRNA. Here, we examined in vivo requirement of stem IIa, stem IIb, stem III and single stranded regions of U12 snRNA using a previously described U12 snRNA mediated branch site mutation suppression assay. Mutations of single stranded linker region immediately following the stem I region revealed that distance constraint between stem I and stem IIa is necessary for splicing. Various mutations of stem IIa including complete deletion of the stem loop were deleterious for in vivo splicing. Surprisingly, mutations of the evolutionarily conserved stem IIb including complete deletion have little or no functional effect. Finally, we show the requirement of stem III terminal loop for in vivo splicing. Interestingly, deletion designed to shorten the length of stem III was functional. Our mutagenesis study of stem III supports the alternative version of the stem loop structure proposed recently. These results show that in vivo sequence requirements of U12 snRNA are similar to those described previously for U2 snRNA and provide experimental support for U12 snRNA intramolecular structure.

Keywords: U12 snRNA, suppression assay, splicing