RRM
2007 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
We previously showed that protein-free fragments of human U6 and U2 snRNAs formed a basepaired complex that resembled the one forming in the activated spliceosomes, and further, provided evidence for splicing-like catalysis in this system. To extend the characterization of the catalytic activity of these two snRNAs, we have started a detailed comparison of the structural features and catalytic activity of U6 and U2 snRNAs derived from human and yeast. The RNAstructure algorithm indicated that while the global base-paired structure of the U6/U2 complex were similar in yeast and human, a number of structural elements, such as U6/U2 helix I and the U2 stemloop I showed a different basepairing pattern. In addition, the conserved ACAGAGA box of U6 was predicted to form a basepaired structure in the yeast U6/U2 complex, while in human this region remains unpaired. To determine the role of these structural differences, we compared the catalytic activity of human and wild type and mutant yeast U6/U2. While these mutations caused very minimal alteration of the primary sequence of yeast U6 and U2, they induced individual domains of the yeast U6/U2 complex to form basepairing interactions similar to those predicted in the human complex. While wild-type and mutant yeast U6/U2 did not differ in substrate binding, catalytic assays indicated that the wild type yeast U6/U2 and all except one of the mutant yeast U6/U2 complexes could not perform splicing-related catalysis in vitro. Intriguingly, the only yeast U6/U2 mutant that showed catalytic activity was the one that resembled the human U6/U2 complex most in terms of its secondary structure. This suggests that the differences in primary sequence of human and yeast U6 and U2 are not incompatible with catalytic activity, and that the secondary structure of the U6/U2 complex makes crucial contributions to the catalytic function of these RNAs.
Keywords: U6U2, Splicing, Catalysis
