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-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 ␣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. ). Polycistronic pre-mRNAs from these operons are processed into monocistronic mRNAs by cleavage and polyadenylation at the 3Ј ends of upstream gene mRNAs accompanied by trans-splicing at the 5Ј ends of downstream gene mRNAs. In general, these two processes occur within a 100-nucleotide region (Blumenthal and Steward 1997) and are mechanistically coupled (Kuersten et al. 1997).In C. elegans, 3Ј end formation is dependent on an AAUAAA signal (Kuersten et al. 1997;Liu et al. 2001). It is expected that this sequence is bound by cleavage and polyadenylation specificity factor (CPSF), as it is in mammalian cells (for reviews, see Colgan and Manley 1997;Keller and Minvielle-Sebastia 1997;Zhao et al. 1999). Presumably, C. elegans 3Ј end formation also requires cleavage stimulation factor (CstF), which binds a U-rich or GU-rich sequence downstream of the cleavage site. Homologs of each of the subunits of both mammalian CstF and CPSF are present in the C. elegans genome (C.J. Wilusz and T. Blumenthal, unpubl.).Trans-splicing generates 5Ј ends of mRNAs in trypanosomes and many animals (Murphy et al. 1986;Sutton and Boothroyd 1986;Krause and Hirsh 1987;Rajkovic et al. 1990;Tessier et al. 1991;Stover and Steele 2001;Vandenberghe et al. 2001). A spliced leader (SL) exon is donated to the 5Ј ends of mRNAs by a short RNA donor called SL RNA. The SL RNA exists as a ribonucleoprotein (RNP) particle (Thomas et al. 1988;Van Doren and Hirsh 1988;Maroney et al. 1990;Goncharov et al. 1999) that includes the Sm core proteins (Lerner and Steitz 1979). Unlike the other U snRNPs, which are capable of catalyzing repeated splicing reactions, the SL snRNP is consumed during the trans-splicing reaction.C. elegans possesses two distinct SL RNAs, SL1 RNA (Krause and Hirsh 1987) a...

An uncapped RNA suggests a model for Caenorhabditis elegans polycistronic pre-mRNA processing

“…CPSF, the protein that recognizes AAUAAA, and CstF are known to interact cooperatively at 3Ј end signals (Gilmartin and Nevins 1991;Murthy and Manley 1992;Wilusz et al 1990). Furthermore, the SL2 snRNP is known to be present in a complex with CstF-64, and this complex has been shown to be important for SL2 trans-splicing (Evans et al 2001). Nonetheless, we have not been able to demonstrate binding of CstF to the Ur element (Y.…”

“…The Ur mutations did not appreciably affect 3Ј end formation , however, and we have been unable to detect an interaction between the Ur element and CstF (unpubl.). On the other hand, the SL2 snRNP has been found in a complex with CstF, and the presence of this complex strongly correlates with the ability of the snRNP to function at operon trans-splice sites (Evans et al 2001). Clearly, CstF might be a protein that acts at the Ur element but it is too soon to conclude that it is.…”

A novel family of C. elegans snRNPs contains proteins associated with trans-splicing

“…Following cleavage catalyzed by the cleavage and polyadenylation specificity factor (CPSF) bound at the AAUAAA just 59 of the cleavage site and cleavage stimulatory factor (CstF) bound to U-rich sequences in the vicinity of the cleavage site (Salisbury et al 2006), the 39 portion of the pre-mRNA is degraded exonucleolytically 59 to 39 until the nuclease reaches a block, possibly a bound protein (Liu et al 2003). CstF has been shown to interact with the SL2 snRNP, and mutations within the third stem-loop of SL2 RNA that eliminate this binding prevent its trans-splicing at operon trans-splice sites (Evans et al 2001). SL RNAs are bound to the Sm proteins that are also core proteins in spliceosomal U1, U2, U4, and U5 snRNPs (Bruzik et al 1988; Thomas et al 1988;Van Doren and Hirsh 1988).…”

Polycistronic pre-mRNA processing in vitro: snRNP and pre-mRNA role reversal in trans-splicing

“…In addition, 39 end formation factor CstF-64 coimmunoprecipitated SL2 RNA but not SL1 RNA. Mutant SL2 RNA sequences that had lost this interaction were unable to trans-splice at downstream operon sites (Evans and Blumenthal 2000;Evans et al 2001). Furthermore, there is a strong rationale for a connection between 39 end formation and trans-splicing.…”

“…SL2 interacts with the 39 end processing factor CstF-64 (Evans et al 2001). This likely provides some of the SL2 (vs. SL1) specificity, since only downstream gene trans-splice sites would be expected to be in proximity to a 39 end formation site.…”