{"doi":"10.5451/unibas-004215005","title":"Unknown","abstract":null,"journal":null,"year":null,"id":648477,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"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
\\nto their export from the nucleus to the cytoplasm. Although all three processing reactions
\\ncan be studied separately in vitro, they are coupled in vivo.
\\n3’ end processing of most mammalian pre-mRNAs involves endonucleolytic
\\ncleavage followed by polyadenylation of the upstream cleavage product. Cleavage and
\\npolyadenylation specificity factor (CPSF) is a multiprotein complex, which together with
\\ncleavage factor Im and IIm (CF Im, CF IIm), cleavage stimulatory factor (CstF), poly(A)
\\npolymerase (PAP) and nuclear poly(A) binding protein 1 (PABPN1) is required for
\\n3’ end formation.
\\nWe have found that the U2 snRNA and subunits of the splicing factors 3a and 3b
\\n(SF3a, SF3b), which are components of the U2 snRNP, were also present in highly
\\npurified CPSF fractions. GST pull-down experiments indicated a direct interaction of
\\nCPSF subunits with SF3b49 and SF3b130. Furthermore, antibodies directed against
\\nCPSF100 co-immunoprecipitated subunits of SF3a and SF3b and the U2 snRNA Taken
\\ntogether our results show that subunits of CPSF and the U2 snRNP directly interact with
\\neach other.
\\nIn order to analyze whether this interaction plays a role in the coupling of 3’ end
\\nprocessing and splicing, we depleted CPSF subunits from HeLa cell nuclear extract and
\\ntested the extracts for splicing activity. CPSF100-depleted extract showed no detectable
\\ncleavage activity and its splicing activity was significantly reduced in coupled assays but
\\nnot in un-coupled assays. Moreover, pre-mRNAs containing mutations in the binding site
\\nof SF3b were not only less efficiently spliced but they also showed reduced cleavage
\\nactivity. Interestingly, efficient cleavage required the presence of the U2 snRNA in
\\ncoupled but not in un-coupled assays. Based on our studies, we propose that the
\\ninteractions between CPSF and U2 snRNP contribute to the coupling of splicing and 3’
\\nend formation.
\\n
\\nFurthermore, we depleted CPSF100 and the U2 snRNP subunits SF3b155 and
\\nSF3b130 by means of RNAi. We observed that knock down of both SF3b proteins caused
\\nhigh lethality of the cells indicating that these polypeptides are essential. However,
\\ndepletion of CPSF100 did not result in a significant increase in cell mortality, suggesting
\\nthat the protein is either not essential that the knock down was not efficient enough to
\\nresult in cell lethality or that CPSF100 shares redundant functions with another protein.
\\nWe were able to show that SF3b155 and SF3b130 are required for efficient splicing in
\\nvivo but did not detect a splicing deficiency in CPSF100 depleted cells. Knock down of
\\nneither of the proteins resulted in an observable 3’ end processing deficiency. Further
\\nwork is required to address the question if the U2 snRNP and CPSF couple splicing and
\\n3’ end processing in vivo.
\\nSplicing and 3’ end formation are highly conserved mechanisms from mammals
\\nto yeast and the two organisms share homologues of most of the proteins involved in the
\\ntwo reactions. To test whether the coupling mechanism mediated by CPSF and the
\\nU2 snRNP is conserved between different organisms, we focused on the yeast system.
\\nThe essential protein Rse1p is the yeast homolog of SF3b130. We show that the rse1-1
\\nstrain is sensitive to cordycepin, which suggests that Rse1p might be involved in 3’ end
\\nprocessing. Furthermore, Rse1p and 3’ end processing factors interacted genetically and
\\nNorthern blot analysis suggested that strains carrying mutations in Rse1p and subunits of
\\nCPF had increased levels of unspliced pre-mRNA at restrictive temperature compared to
\\nthe single mutants. We therefore suggest that the coupling of 3’ end processing and
\\nsplicing mediated by CPSF and U2 snRNP is conserved between mammals and yeast.
\\nPrecursor tRNAs (pre-tRNAs) must undergo a number of processing steps before
\\nthey become mature tRNAs and some tRNAs contain introns. tRNA splicing is a three
\\nstep reaction and each step requires an individual set of proteins. In the first step the pretRNA
\\nis cut at its two splice sites. This reaction is catalyzed by the so called tRNA
\\nsplicing endonuclease complex. Recently this complex was purified from mammalian
\\ncells and interestingly hClp1 (a subunit of the 3’ end processing factor CF IIm) was
\\nidentified as one of its components. Furthermore, hSen2 a subunit of the endonuclease
\\ncomplex was shown to be required for efficient 3’ end processing in vivo. A model was
\\nproposed suggesting that the tRNA endonuclease complex is involved in 3’ end
\\n
\\nprocessing. In collaboration with S. Paushkin and C. Trotta we continued to investigate if
\\nthis model is indeed correct. We found that biochemically purified CF IIm indeed carried
\\ntRNA endonuclease activity. However, tRNA endonuclease complexes were not able to
\\nreconstitute cleavage activity of CF IIm-depleted HeLa nuclear extract, unless they were
\\npurified with His-Flag tagged hClp1. Taken all our results into account we cannot
\\nexclude that the tRNA endonuclease complex is indeed involved in 3’ end processing.
\\nHowever, we believe that the evidence supporting this model is rather weak. We think it
\\nis more likely that hClp1 is part of the tRNA endonuclease complex as well as a subunit
\\nof CF IIm, and that the two complexes are not functionally related.
\\nAs mentioned earlier, 3’ end processing is highly conserved form mammals to
\\nyeast. In S. cerevisiae cleavage and polyadenylation factor (CPF) is a multiprotein
\\ncomplex, which together with the cleavage factor IA (CF IA) and the cleavage factor IB
\\n(CF IB) is required for both the cleavage and the polyadenylation step of the 3’ end
\\nformation reaction.
\\nYdh1p/Cft2p is an essential component of CPF. Cleavage and polyadenylation
\\nreactions revealed that the protein is required for both reactions to occur in vitro.
\\nPreviously, it was demonstrated that an important function of CPF lies in the recognition
\\nof poly(A) site sequences and previous RNA binding analyses with recombinant
\\nYdh1p/Cft2p suggested that the protein may interact with the CYC1 poly(A) site region.
\\nIn accordance, we found that mutant ydh1 strains were deficient in recognition of the
\\nACT1 cleavage site in vivo.
\\nTranscription by RNA polymerase II (RNAP II) and 3’ end processing reactions
\\nare tightly linked. The C-terminal domain (CTD) of RNAP II plays a major role in
\\ncoupling the two events, as it tethers the factors involved in polyadenylation to the
\\npolymerase. We provide evidence that Ydh1p/Cft2p interacts with the CTD, several
\\nsubunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p
\\ncontributes to the formation of important interaction surfaces that mediate the dynamic
\\nassociation of CPF with RNAP II, the recognition of poly(A) site sequences and the
\\nassembly of the polyadenylation machinery on the RNA substrate.","is_dataset_classified":null,"base_score":0.0,"endowment":0.0,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"19965766","pmcid":null,"openalex_id":"https://openalex.org/W1601072800","authors":[],"funders":[],"total_grants":0,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[],"oa_status":"green","license":null,"oa_locations":[{"url":"https://doi.org/10.5451/unibas-004215005","host_type":"repository"},{"url":"https://edoc.unibas.ch/596/","host_type":""}],"fields_of_study":["RNA Research and Splicing","RNA and protein synthesis mechanisms","Neurogenetic and Muscular Disorders Research"],"mesh_terms":[],"keywords":["snRNP","Cleavage and polyadenylation specificity factor","Cleavage factor","Precursor mRNA","RNA splicing","Polyadenylation","Cleavage stimulation factor","Cell biology","Biology","Cleavage (geology)","RNA-binding protein","Small nuclear ribonucleoprotein","Small nuclear RNA","Immunoprecipitation","Molecular biology","Messenger RNA","Biochemistry","RNA","Gene"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-10T02:39:13.321978Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}