Reconstitution of 3′ end processing of mammalian pre-mRNA reveals a central role of RBBP6
- Moritz Schmidt1,5,
- Florian Kluge1,5,
- Felix Sandmeir2,5,
- Uwe Kühn1,5,
- Peter Schäfer1,
- Christian Tüting1,4,
- Christian Ihling3,
- Elena Conti2 and
- Elmar Wahle1
- 1Institute of Biochemistry and Biotechnology, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany;
- 2Department of Structural Cell Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany;
- 3Institute of Pharmacy, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany
- Corresponding authors: ewahle{at}biochemtech.uni-halle.de, conti{at}biochem.mpg.de
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↵5 These authors contributed equally to this work.
Abstract
The 3′ ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.
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Footnotes
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Supplemental material is available for this article.
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Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.349217.121.
- Received November 17, 2021.
- Accepted January 21, 2022.
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