The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation.
Cleavage-polyadenylation specificity factor (CPSF) is a multisubunit protein that plays a central role in 3' processing of mammalian pre-mRNAs. CPSF recognizes the AAUAAA signal in the pre-mRNA and interacts with other proteins to facilitate both RNA cleavage and poly(A) synthesis. Here we describe the isolation of cDNAs encoding the largest subunit of CPSF (160K) as well as characterization of the protein product. Antibodies raised against the recombinant protein inhibit polyadenylation in vitro, which can be restored by purified CPSF. Extending previous studies, which suggested that 160K contacts the pre-mRNA, we show that purified recombinant 160K can, by itself, bind preferentially to AAUAAA-containing RNAs. While the sequence of 160K reveals similarities to the RNP1 and RNP2 motifs found in many RNA-binding proteins, no clear 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) polymerase (PAP). These results provide explanations for previously observed cooperative interactions between CPSF and CstF, which are responsible for poly(A) site specification, and between CPSF and PAP, which are necessary for synthesis of the poly(A) tail. Also supporting a direct role for 160K in these interactions is the fact that 160K by itself retains partial ability to cooperate with CstF in binding pre-mRNA and, unexpectedly, inhibits PAP activity in in vitro assays. We discuss the significance of these multiple functions and also a possible evolutionary link between yeast and mammalian polyadenylation suggested by the properties and sequence of 160K.
Murthy
K G
KG
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Manley
J L
JL
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors
D018076:DNA, Complementary; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D011061:Poly A; D011062:Polynucleotide Adenylyltransferase