THE 160-KD SUBUNIT OF HUMAN CLEAVAGE POLYADENYLATION SPECIFICITY FACTOR COORDINATES PRE-MESSENGER-RNA 3'-END FORMATION

Cleavage-polyadenylation specificity factor (CPSF) is a multisubunit p rotein that plays a central role in 3' processing of mammalian pre-mRN As. CPSE recognizes the AAUAAA signal in the pre-mRNA and interacts wi th other proteins to facilitate both RNA cleavage and poly(A) synthesi s. Here we describe the isolation of cDNAs encoding the largest subuni t of CPSE (160K) as well as characterization of the protein product. A ntibodies raised against the recombinant protein inhibit polyadenylati on in vitro, which can be restored by purified CPSF. Extending previou s studies, which suggested that 160K contacts the pre-mRNA, we show th at purified recombinant 160K can, by itself, bind preferentially to AA UAAA-containing RNAs. While the sequence of 160K reveals similarities to the RNP1 and RNP2 motifs found in many RNA-binding proteins, no cle ar match to a known RNA-binding domain was found, and RNA recognition is therefore likely mediated by a highly diverged or novel structure. We also show that 160K binds specifically to both the 77K (suppressor of forked) subunit of the cleavage factor CstF and to poly(A) polymera se (PAP). These results provide explanations for previously observed c ooperative interactions between CPSF and CstF, which are responsible f or poly(A) site specification, and between CPSF and PAP, which are nec essary for synthesis of the poly(A) tail. Also supporting a direct rol e for 160K in these interactions is the fact that 160K by itself retai ns partial ability to cooperate with CstF in binding pre-mRNA and, une xpectedly, inhibits PAP activity in in vitro assays. We discuss the si gnificance of these multiple functions and also a possible evolutionar y link between yeast and mammalian polyadenylation suggested by the pr operties and sequence of 160K.