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Recent Insights into the Regulation of mRNA Splicing
Author(s) -
Guthrie Christine
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.90.1
Subject(s) - spliceosome , snrnp , small nuclear rna , rna splicing , prp24 , intron , rna , biology , microbiology and biotechnology , genetics , minor spliceosome , gene , non coding rna
The spliceosome is a highly dynamic RNA‐protein machine requiring the ordered binding and release of 5 small nuclear ribonucleoprotein particles (snRNPs) to the intron‐containing pre‐mRNA. Following the recognition of the 5' splice site by base‐pairing to U1 snRNA and the intron branchpoint sequence by base‐pairing to U2 snRNA, the U4/U6.U5 triple snRNP joins the assembling spliceosome. In this complex, U4 snRNA is extensively base‐paired to U6 snRNA. Yet despite the presence of all 5 essential snRNAs, this form of the spliceosome is catalytically inactive. The 2 chemical steps require the unwinding of U4 from U6 and the coordinated release of U1 snRNP. This strategy presumably ensures that catalytic activation is restricted until the appropriate intronic signals have been accurately identified. We are using a combination of biochemical and genetic approaches to understand the mechanistic underpinnings of this fidelity mechanism. Key players are Brr2, an RNA‐dependent ATPase, Snu114, an EF2‐like GTPase, and Prp8, the largest and most evolutionarily conserved spliceosomal protein. Brr2 is required for the ATP‐dependent unwinding of U4/U6 and we have recently recapitulated this reaction in a minimal in vitro system using synthetic RNAs. Notably, this reaction requires the presence of a C‐terminal fragment of Prp8 and this stimulation is inhibited by mutations that give rise to retinitis pigmentosa in humans. Since Prp8 and Brr2 are both constitutive components of the spliceosome, we now seek to understand how the productive interaction of the C‐terminus of Prp8 with Brr2 is itself regulated. An interesting hypothesis is that this requires the GTP‐dependent conformational rearrangement of Snu114 with respect to Prp8.