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-specific trans-splicing of the downstream mRNA. These processes usually occur within an approximately 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 SL2 snRNP. This complex, immunoprecipitated with alphaCstF-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 a trans-splice donor, but fails to splice to SL2-accepting trans-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 for trans-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.
Evans
D
D
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Denver, CO 80262, USA.
Perez
I
I
MacMorris
M
M
Leake
D
D
Wilusz
C J
CJ
Blumenthal
T
T
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D017931:DNA Primers; D009154:Mutation; D009876:Operon; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020038:RNA, Spliced Leader; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors
D017931:DNA Primers; D012333:RNA, Messenger; D020038:RNA, Spliced Leader; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D039221:mRNA Cleavage and Polyadenylation Factors