A complex containing CstF-64 and the SL2 snRNP connects mRNA 3 ' end formation and trans-splicing in C-elegans operons

Polycistronic pre-mRNAs from Caenorhabditis elegans are processed by 3' end formation of the upstream mRNA and SL2-specffic trans-splicing of the down stream mRNA. These processes usually occur within an -100-nucleotide region and are mechanistically coupled. In this paper, we report a complex in C. elegans extracts containing the 3' end formation protein CstF-64 and the SL 2 snRNP. This complex, immunoprecipitated,with alpha CstF-64 antibody, cont ains SL2 RNA, but not SL1 RNA or other U snRNAs. Using mutational analysis we have been able to uncouple SL2 snRNP function and identity. SL2 RNA with a mutation in stem/loop III is functional in vivo as a trans-splice donor, but fails to splice to SL2-accepting trans-splice sites, suggesting that i t has lost its identity as an SL2 snRNP. Importantly, stem/loop III mutatio ns prevent association of SL2 RNA with CstF-64. In contrast, a mutation in stem II that inactivates the SL2 snRNP still permits complex formation with CstF-64. Therefore, SL2 RNA stem/loop III is required for both SL2 identit y and formation of a complex containing CstF-64, but not for trans-splicing . These results provide a molecular framework for the coupling of 3' end fo rmation and trans-splicing in the processing of polycistronic pre-mRNAs fro m C. elegans operons.