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Genes Dev. 2014 Nov 1;28(21):2381-93. doi: 10.1101/gad.250985.114. Epub 2014 Oct 9.

Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33.

Author information

1
Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany;
2
Computational and Systems Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
3
Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany; ewahle@biochemtech.uni-halle.de.

Abstract

Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.

KEYWORDS:

3′ end formation; RNA processing; poly(A) polymerase; poly(A) site; polyadenylation

PMID:
25301781
PMCID:
PMC4215183
DOI:
10.1101/gad.250985.114
[Indexed for MEDLINE]
Free PMC Article

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