RRM
2009 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Determining the identity of the catalytic domain of the spliceosome has been a central question in the splicing field for the last two decades. Experimental evidence in vivo and in cellular extracts have indicated that a basepaired complex of U6 and U2 snRNAs in the activated spliceosomes plays a critical role in catalysis of the splicing reaction. In addition, mechanistic and structural similarities to the self-splicing group II introns has led to the widely-believed hypothesis that the spliceosomal RNAs probably form the active site of the splicing reaction, with the spliceosomal proteins playing regulatory and supporting roles. In order to understand the mechanism of how the spliceosomal RNAs affect catalysis, we have successfully reconstituted the catalytic heart of the spliceosome from scratch by assembling the base-paired complex of U6 and U2 snRNAs in vitro. Using in vitro-synthesized, protein free human U6 and U2 snRNAs, we succeeded in forming a U6/U2 base-paired complex that closely resembled the one observed in the activated spliceosomes in terms of its secondary and three-dimensional structure. Intriguingly, we have recently showed that the U6/U2 complex can indeed catalyze a two step reaction resembling the first and second steps of splicing on a pre-mRNA model construct (Valadkhan et al., 2009), thus performing the full spliceosomal catalytic cycle without the help of any proteins in vitro. Using this minimal splicing system, we have studied the effect of several mutations, which disrupt interactions between critical active site elements, on splicing pattern. Interestingly, insertion and deletion mutations in Helix II and Stem I of U2, did not affect the catalytic activity. Even when the whole Helix II and the whole StemI of U2 were deleted, the catalytic activity was unaffected. However, when there were insertion and deletion mutations in the ISL (Intramolecular Stemloop) and Helix I region, we have observed aberrant spliced products. The severity of aberrant splicing was increased when there were mutations in both lower ISL and Helix I. Work is currently underway to determine the effect of similar mutations on splicing in vivo using a orthogonal system in HeLa cells. Taken together these data will help us define the mechanism of splice site selection and the role of snRNAs in specifiying the splice site.
Keywords: U6U2, Splicing
