Characterization of factors involved in the coupling of 3' end processing and splicing and in the 3' end formation of mRNA precursors

Abstract

Eukaryotic mRNA precursors are processed at their 5’ and 3’ ends and are spliced prior to their export from the nucleus to the cytoplasm. Although all three processing reactions can be studied separately in vitro, they are coupled in vivo. 3’ end processing of most mammalian pre-mRNAs involves endonucleolytic cleavage followed by polyadenylation of the upstream cleavage product. Cleavage and polyadenylation specificity factor (CPSF) is a multiprotein complex, which together with cleavage factor Im and IIm (CF Im, CF IIm), cleavage stimulatory factor (CstF), poly(A) polymerase (PAP) and nuclear poly(A) binding protein 1 (PABPN1) is required for 3’ end formation. We have found that the U2 snRNA and subunits of the splicing factors 3a and 3b (SF3a, SF3b), which are components of the U2 snRNP, were also present in highly purified CPSF fractions. GST pull-down experiments indicated a direct interaction of CPSF subunits with SF3b49 and SF3b130. Furthermore, antibodies directed against CPSF100 co-immunoprecipitated subunits of SF3a and SF3b and the U2 snRNA Taken together our results show that subunits of CPSF and the U2 snRNP directly interact with each other. In order to analyze whether this interaction plays a role in the coupling of 3’ end processing and splicing, we depleted CPSF subunits from HeLa cell nuclear extract and tested the extracts for splicing activity. CPSF100-depleted extract showed no detectable cleavage activity and its splicing activity was significantly reduced in coupled assays but not in un-coupled assays. Moreover, pre-mRNAs containing mutations in the binding site of SF3b were not only less efficiently spliced but they also showed reduced cleavage activity. Interestingly, efficient cleavage required the presence of the U2 snRNA in coupled but not in un-coupled assays. Based on our studies, we propose that the interactions between CPSF and U2 snRNP contribute to the coupling of splicing and 3’ end formation. Furthermore, we depleted CPSF100 and the U2 snRNP subunits SF3b155 and SF3b130 by means of RNAi. We observed that knock down of both SF3b proteins caused high lethality of the cells indicating that these polypeptides are essential. However, depletion of CPSF100 did not result in a significant increase in cell mortality, suggesting that the protein is either not essential that the knock down was not efficient enough to result in cell lethality or that CPSF100 shares redundant functions with another protein. We were able to show that SF3b155 and SF3b130 are required for efficient splicing in vivo but did not detect a splicing deficiency in CPSF100 depleted cells. Knock down of neither of the proteins resulted in an observable 3’ end processing deficiency. Further work is required to address the question if the U2 snRNP and CPSF couple splicing and 3’ end processing in vivo. Splicing and 3’ end formation are highly conserved mechanisms from mammals to yeast and the two organisms share homologues of most of the proteins involved in the two reactions. To test whether the coupling mechanism mediated by CPSF and the U2 snRNP is conserved between different organisms, we focused on the yeast system. The essential protein Rse1p is the yeast homolog of SF3b130. We show that the rse1-1 strain is sensitive to cordycepin, which suggests that Rse1p might be involved in 3’ end processing. Furthermore, Rse1p and 3’ end processing factors interacted genetically and Northern blot analysis suggested that strains carrying mutations in Rse1p and subunits of CPF had increased levels of unspliced pre-mRNA at restrictive temperature compared to the single mutants. We therefore suggest that the coupling of 3’ end processing and splicing mediated by CPSF and U2 snRNP is conserved between mammals and yeast. Precursor tRNAs (pre-tRNAs) must undergo a number of processing steps before they become mature tRNAs and some tRNAs contain introns. tRNA splicing is a three step reaction and each step requires an individual set of proteins. In the first step the pretRNA is cut at its two splice sites. This reaction is catalyzed by the so called tRNA splicing endonuclease complex. Recently this complex was purified from mammalian cells and interestingly hClp1 (a subunit of the 3’ end processing factor CF IIm) was identified as one of its components. Furthermore, hSen2 a subunit of the endonuclease complex was shown to be required for efficient 3’ end processing in vivo. A model was proposed suggesting that the tRNA endonuclease complex is involved in 3’ end processing. In collaboration with S. Paushkin and C. Trotta we continued to investigate if this model is indeed correct. We found that biochemically purified CF IIm indeed carried tRNA endonuclease activity. However, tRNA endonuclease complexes were not able to reconstitute cleavage activity of CF IIm-depleted HeLa nuclear extract, unless they were purified with His-Flag tagged hClp1. Taken all our results into account we cannot exclude that the tRNA endonuclease complex is indeed involved in 3’ end processing. However, we believe that the evidence supporting this model is rather weak. We think it is more likely that hClp1 is part of the tRNA endonuclease complex as well as a subunit of CF IIm, and that the two complexes are not functionally related. As mentioned earlier, 3’ end processing is highly conserved form mammals to yeast. In S. cerevisiae cleavage and polyadenylation factor (CPF) is a multiprotein complex, which together with the cleavage factor IA (CF IA) and the cleavage factor IB (CF IB) is required for both the cleavage and the polyadenylation step of the 3’ end formation reaction. Ydh1p/Cft2p is an essential component of CPF. Cleavage and polyadenylation reactions revealed that the protein is required for both reactions to occur in vitro. Previously, it was demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and previous RNA binding analyses with recombinant Ydh1p/Cft2p suggested that the protein may interact with the CYC1 poly(A) site region. In accordance, we found that mutant ydh1 strains were deficient in recognition of the ACT1 cleavage site in vivo. Transcription by RNA polymerase II (RNAP II) and 3’ end processing reactions are tightly linked. The C-terminal domain (CTD) of RNAP II plays a major role in coupling the two events, as it tethers the factors involved in polyadenylation to the polymerase. We provide evidence that Ydh1p/Cft2p interacts with the CTD, several subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.