Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit.
During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.
Department of Cell Biology, University of Basel, Switzerland.
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Hauri
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Keller
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D039221:mRNA Cleavage and Polyadenylation Factors
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors