Purification and Characterization of Human Cleavage Factor I Involved in the 3′ End Processing of Messenger RNA Precursors (*)

Six different protein factors are required for the specific cleavage and polyadenylation of pre-mRNA in mammals. Whereas four of them have been purified and most of their components cloned, cleavage factor I (CF I) and cleavage factor II (CF II) remained poorly characterized. We report here the separation of CF I from CF II and the purification of CF I to near homogeneity. Three polypeptides of 68, 59, and 25 kDa copurify with CF I activity. All three polypeptides can be UV cross-linked to a cleavage and polyadenylation substrate in the presence of a large excess of unspecific competitor RNA, but not to a splicing-only substrate. No additional protein factor is required for the binding of CF I to pre-mRNA. Gel retardation experiments confirmed the results obtained by UV cross-linking. In addition, we could show that CF I stabilizes the binding of the cleavage and polyadenylation specificity factor (CPSF) to pre-mRNA and that CPSF and CF I together form a slower migrating complex with pre-mRNA than the single protein factors. Cleavage stimulation factor (CstF) and poly(A) polymerase (PAP) had no detectable effect on the binding of CF I to pre-mRNA. Furthermore, the CstF•CPSF•RNA as well as the CstF•CPSF• PAP•RNA complex are supershifted and stabilized upon the addition of CF I.

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