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

Abstract

Polycistronic pre-mRNAs from Caenorhabditis elegans are processed by 3′ end formation of the upstream mRNA and SL2-specifictrans-splicing of the downstream mRNA. These processes usually occur within an ∼100-nucleotide region and are mechanistically coupled. In this paper, we report a complex in C. elegansextracts containing the 3′ end formation protein CstF-64 and the SL2 snRNP. This complex, immunoprecipitated with αCstF-64 antibody, contains 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 atrans-splice donor, but fails to splice to SL2-acceptingtrans-splice sites, suggesting that it has lost its identity as an SL2 snRNP. Importantly, stem/loop III mutations 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 identity and formation of a complex containing CstF-64, but not fortrans-splicing. These results provide a molecular framework for the coupling of 3′ end formation and trans-splicing in the processing of polycistronic pre-mRNAs from C. elegans operons.