Reconstitution of mammalian cleavage factor II involved in 3′ processing of mRNA precursors
- Peter Schäfer1,
- Christian Tüting1,
- Lars Schönemann1,5,
- Uwe Kühn1,
- Thomas Treiber2,
- Nora Treiber2,
- Christian Ihling3,
- Anne Graber1,4,6,
- Walter Keller4,
- Gunter Meister2,
- Andrea Sinz3 and
- Elmar Wahle1
- 1Institute of Biochemistry and Biotechnology, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany
- 2Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany
- 3Institute of Pharmacy, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany
- 4Biozentrum, University of Basel, CH-4056 Basel, Switzerland
- Corresponding author: ewahle{at}biochemtech.uni-halle.de
Abstract
Cleavage factor II (CF II) is a poorly characterized component of the multiprotein complex catalyzing 3′ cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5′ kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.068056.118.
- Received July 9, 2018.
- Accepted August 17, 2018.
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