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PMID:7590244

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Citation

Murthy, KG and Manley, JL (1995) The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation. Genes Dev. 9:2672-83

Abstract

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.

Links

PubMed

Keywords

Amino Acid Sequence; Base Sequence; DNA, Complementary; Humans; Molecular Sequence Data; Poly A/genetics; Poly A/metabolism; Polynucleotide Adenylyltransferase/metabolism; RNA Precursors/chemistry; RNA-Binding Proteins/chemistry; RNA-Binding Proteins/genetics; RNA-Binding Proteins/metabolism; mRNA Cleavage and Polyadenylation Factors

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:PAPOA

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q10570

F

Seeded From UniProt

complete

HUMAN:CPSF1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q12996

F

Seeded From UniProt

complete

HUMAN:CPSF1

enables

GO:0019899: enzyme binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P51003

F

Seeded From UniProt

complete

HUMAN:CPSF1

involved_in

GO:0006378: mRNA polyadenylation

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:CPSF1

part_of

GO:0005847: mRNA cleavage and polyadenylation specificity factor complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:CPSF1

enables

GO:0035925: mRNA 3'-UTR AU-rich region binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:CPSF1

involved_in

GO:0098789: pre-mRNA cleavage required for polyadenylation

ECO:0000305: curator inference used in manual assertion

GO:0005847

P

Seeded From UniProt

complete

HUMAN:CSTF3

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q10570

F

Seeded From UniProt

complete

HUMAN:PAPOA

enables

GO:0004652: polynucleotide adenylyltransferase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:PAPOA

involved_in

GO:0006378: mRNA polyadenylation

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

Notes

See also

References

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