Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus.
Cleavage-polyadenylation specificity factor (CPSF) is one of five separable factors known to be required for 3' cleavage and polyadenylation of mRNA precursors in vitro. Previous studies have shown that the cleavage and poly(A) addition reactions can be uncoupled in vitro and have suggested that CPSF may be the only factor essential for both of these subreactions. Here we report the purification of CPSF to near homogeneity from calf thymus and show that the purified factor contains three polypeptides of 165, 105, and 70 kDa. These polypeptides cosediment precisely with CPSF activity, which has a sedimentation coefficient of 11.5 S. Consistent with previous reports from our laboratory, purified CPSF does not contain a detectable RNA component, indicating that it is a multisubunit protein and not a small nuclear ribonucleoprotein. Extensively purified bovine CPSF can function with human poly(A) polymerase to bring about AAUAAA-dependent poly(A) addition or with human cleavage factors to catalyze accurate 3' cleavage of a pre-mRNA substrate. UV cross-linking and gel retention analyses demonstrate that highly purified CPSF interacts with one of these cleavage factors, the multisubunit cleavage-stimulation factor, to facilitate stable binding of both to an AAUAAA-containing pre-mRNA. Likewise, evidence is presented indicating that poly(A) polymerase and CPSF can interact directly.
Murthy
K G
KG
Department of Biological Science, Columbia University, New York, New York 10027.
Manley
J L
JL
D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013950:Thymus Gland / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors
D012322:RNA Precursors; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors