Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA

Abstract

Poly(A) site processing of a pre-mRNA requires the participation of multiple nuclear factors. Two of these factors recognize specific sequences in the pre-mRNA and form a stable processing complex. Since these initial interactions are likely critical for the recognition of the poly(A) site and the efficiency of poly(A) site use, we have characterized these factors and the nature of their interaction with the pre-mRNA. The AAUAAA specificity factor PF2 is a large, multicomponent complex composed of at least five distinct polypeptides ranging in molecular size from 170 to 42 kDa. The 170-kDa polypeptide appears to mediate interaction with the pre-mRNA. Factor CF1, which provides specificity for the downstream G + U-rich element and stabilizes the PF2 interaction on the RNA, is also a multicomponent complex but is less complex than PF2. CF1 is composed of three polypeptides of molecular sizes 76, 64, and 48 kDa. UV cross-linking assays demonstrate that the 64-kDa polypeptide makes direct contact with the RNA, dependent on the G + U-rich downstream sequence element. Moreover, it is clear that these RNA-protein interactions are influenced by the apparent cooperative interaction involving PF2 and CF1, interactions that contribute to the efficiency of poly(A) site processing.