Saha D, Khandelia P, O'Keefe RT, Vijayraghavan U

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation is critical for splice site selection and catalysis. The highly conserved NTC protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NTC associated proteins is the non-essential protein Bud31/Ycr063w, which is also a component ...

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of the Cef1p sub-complex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed its earliest association is with pre-catalytic B complex and the interaction continues in catalytically active complexes with stably bound U2, U5 and U6 snRNPs. In complementary experiments, wherein pre-catalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Crosslinking of proteins to pre-mRNAs with a site-specific 4-thio Uridine residue at -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a ~25kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.

Date: Jan. 03, 2012

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