Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33
- Lars Schönemann1,
- Uwe Kühn1,
- Georges Martin2,
- Peter Schäfer1,
- Andreas R. Gruber2,
- Walter Keller2,
- Mihaela Zavolan2 and
- Elmar Wahle1
- 1Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany;
- 2Computational and Systems Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland
- Corresponding author: ewahle{at}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
Footnotes
-
Supplemental material is available for this article.
-
Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.250985.114.
- Received August 13, 2014.
- Accepted September 23, 2014.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
