A complex containing CstF-64 and the SL2 snRNP connects mRNA 3' end formation and trans-splicing in C. elegans operons - PubMed (original) (raw)
A complex containing CstF-64 and the SL2 snRNP connects mRNA 3' end formation and trans-splicing in C. elegans operons
D Evans et al. Genes Dev. 2001.
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 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.
Figures
Figure 1
SL2 RNA is specifically immunoprecipitated by αCstF64. (A) U snRNPs were immunoprecipitated from a wild-type C. elegans crude embryonic extract with an antibody raised against the C. elegans homolog of the mammalian 64-kD subunit of the 3′ end formation factor CstF, as well as a pre-immune control antibody. RNA was extracted and analyzed by primer extension with a pool of oligonucleotides, each specific for one of the snRNAs. The oligonucleotides were designed to result in termination at different locations on the gel as indicated. The unlabeled bands below U2 represent U2 breakdown products (data not shown). (T) Total before immunoprecipitation (IP); (PI) preimmune control IP; (CstF) αCstF-64 IP. (B) The results from A were quantified using a Molecular Dynamics PhosphorImager. The values shown are the percent of the total of each RNA immunoprecipitated after subtracting the signal from the pre-immune control.
Figure 2
SL2 RNA mutations still allow core snRNP formation. (A) The mutations studied are shown (boxed and in boldface type) adjacent to the predicted SL2α RNA structure. Nucleotide positions that are conserved in 100% of known SL2 RNA genes (boxed and shaded; Evans et al. 1997) are shown. The _trans_-splice site is marked by an arrow. The stems are numbered as they are referred to in the text and the Sm-binding site is indicated. (B) snRNPs were immunoprecipitated from wild-type or transgenic C. elegans crude embryonic extracts with a human anti-Sm serum. The RNAs contained in the unbound (U), washes (W1-W3), and bound (B) fractions were analyzed by primer extension in the presence of ddCTP to analyze SL2 RNA and by Northern blotting for the controls. The Northern blot was hybridized to a U1 snRNA probe as a positive control, stripped, and hybridized to a 5S rRNA probe as a negative control.
Figure 3
Mutation of the third stem/loop prevents interaction of the SL2 snRNP with CstF64. (A) Immunoprecipitations of C. elegans crude embryonic extracts from untransformed or transgenic strains were performed with αCstF-64 and non-immune control serum. The presence of the SL1 and SL2 RNAs was determined by primer extension in the presence of ddCTP. (B) Quantification of the results shown in A. The values are the percent of the total signal bound to the CstF beads after subtraction of the non-immune control.
Figure 4
rrs-1 rescue by transgenic SL2. (A) Percentage of transgenic (GFP+) progeny that are dead embryos (open bars) or stunted larvae (shaded bars) are shown for each stable transgenic line. The total number of GFP+ progeny scored (n) is indicated for each line below the bar. (B) Rescue efficiency for each set of transgenic lines in A is presented as a fraction, where the rescue efficiency is calculated as an average (with standard deviation shown) from the number of stunted larvae divided by the 25% expected for 100% rescue of the rrs-1unc-76/rrs-1 unc-76 embryonic lethal class.
Figure 5
A two-step model for polycistronic pre-mRNA processing. In the first step, CPSF and CstF collaborate to cleave the pre-mRNA between the two binding sites, as in vertebrate 3′ end formation. CPSF is bound to the AAUAAA and CstF is bound to the U-rich region just downstream. This region, called Ur, has been shown to be required for SL2-specific _trans_-splicing (Huang et al. 2001). In the second step, the mature 5′ end of the downstream mRNA is generated by _trans_-splicing with the SL2 snRNP, delivered to the nearby _trans_-splice site via its interaction with CstF. For simplicity, competing SL1 RNPs are not shown, however, SL1 RNA is seven- to 10-fold more abundant than SL2 RNA. The model illustrates a direct interaction between CstF and the SL2 snRNP. However, it remains possible that the interaction is indirect and is bridged by an unidentified factor.
References
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