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 polyaden ylation specificity factor (CPSF) is required for 3' cleavage of the t ranscript as well as for subsequent polyadenylation. Using peptide seq uences from a tryptic digest, we have cloned the 100-kDa subunit of CP SF. This subunit is a novel protein shelving no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and m onoclonal antibodies were used to characterize the composition of CPSF . Immunoprecipitations of CPSF from HeLa cell extracts and from labele d chromatographic fractions show the coprecipitation of all four subun its of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are s pecifically cross-linked to precursor RNA by UV irradiation were ident ified as CPSF subunits by immunoprecipitation. Immunofluorescent detec tion of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.