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Vol. 15, No. 19, pp. 2562-2571, October 1, 2001

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

Donald Evans,1 Ismael Perez, Margaret MacMorris, Devin Leake, Carol J. Wilusz,2 and Thomas Blumenthal3

Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Denver, Colorado 80262, USA

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 ~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 alpha CstF-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.

[Key Words: Spliced leader RNA; polycistronic transcription; cleavage; polyadenylation]

Present addresses: 1MCDB, University of Colorado, Boulder, CO 80309, USA; 2Deptartment of Molecular Genetics and Microbiology, UMDNJ, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

3 Corresponding author.

GENES & DEVELOPMENT 15:2562-2571 © 2001 by Cold Spring Harbor Laboratory Press  ISSN 0890-9369/01 $5.00
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