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      Composition and processing activity of a semi-recombinant holo U7 snRNP

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          Abstract

          In animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3′ end by U7 snRNP consisting of two core components: a ∼60-nucleotide U7 snRNA and a ring of seven proteins, with Lsm10 and Lsm11 replacing the spliceosomal SmD1 and SmD2. Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7 snRNP. Here, we assembled core U7 snRNP bound to FLASH from recombinant components and analyzed its appearance by electron microscopy and ability to support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm ring assembles efficiently in vitro on a spliceosomal Sm site but the engineered U7 snRNP is functionally impaired. This approach offers a unique opportunity to study the importance of various regions in the Sm proteins and U7 snRNA in 3′ end processing of histone pre-mRNAs.

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          Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis.

          J. Zhao, L. Hyman, C. Moore (1999)
          Formation of mRNA 3' ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3' ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.
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            Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.

            David Mandel, Liang Tong, Vasupradha Vethantham … (2006)
            Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3'-end. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known. Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 A resolution, complexed with zinc ions and a sulphate that might mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1) at 2.5 A resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-beta-lactamase domain and a novel beta-CASP (named for metallo-beta-lactamase, CPSF, Artemis, Snm1, Pso2) domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.
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              Protein factors in pre-mRNA 3'-end processing.

              David Mandel, Liang Tong, Y. Bai (2008)
              Most eukaryotic mRNA precursors (premRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3'-end. Processing at the 3'-end is controlled by sequence elements in the pre-mRNA (cis elements) as well as protein factors. Despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. The 3'-end processing machinery also has important roles in transcription and splicing. The mammalian machinery contains several sub-complexes, including cleavage and polyadenylation specificity factor, cleavage stimulation factor, cleavage factor I, and cleavage factor II. Additional protein factors include poly(A) polymerase, poly(A)-binding protein, symplekin, and the C-terminal domain of RNA polymerase II largest subunit. The yeast machinery includes cleavage factor IA, cleavage factor IB, and cleavage and polyadenylation factor.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Nucleic Acids Res
                Journal ID (iso-abbrev): Nucleic Acids Res
                Journal ID (publisher-id): nar
                Title: Nucleic Acids Research
                Publisher: Oxford University Press
                ISSN (Print): 0305-1048
                ISSN (Electronic): 1362-4962
                Publication date (Print): 20 February 2020
                Publication date (Electronic): 10 December 2019
                Publication date PMC-release: 10 December 2019
                Volume: 48
                Issue: 3
                Pages: 1508-1530
                Affiliations
                [1 ] Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA
                [2 ] Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences , 02-106 Warsaw, Poland
                [3 ] Department of Biological Sciences, Columbia University , New York, NY 10027, USA
                [4 ] California NanoSystems Institute, University of California Los Angeles , Los Angeles, CA 90095, USA
                [5 ] Institute of Genetics and Biotechnology, Warsaw University , 02-106 Warsaw, Poland
                [6 ] Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA
                Author notes
                To whom correspondence should be addressed. Tel: +1 919 962 2139; Fax: +1 919 962 2139; Email: dominski@ 123456med.unc.edu
                Correspondence may also be addressed to Liang Tong. Email: ltong@ 123456columbia.edu

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.

                Author information
                http://orcid.org/0000-0002-0563-6468
                Article
                Publisher ID: gkz1148
                DOI: 10.1093/nar/gkz1148
                PMC ID: 7026596
                PubMed ID: 31819999
                SO-VID: f680b89b-e7bb-477e-a623-72c47df0fd70
                Copyright © © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date accepted : 25 November 2019
                Date revision received : 29 October 2019
                Date received : 31 May 2019
                Page count
                Pages: 23
                Funding
                Funded by: National Institutes of Health 10.13039/100000002
                Award ID: GM 29832
                Award ID: R35GM118093
                Funded by: Polish National Science Centre 10.13039/501100004281
                Award ID: UMO-2014/14/A/NZ1/00306
                Categories
                Subject: RNA and RNA-protein complexes

                ScienceOpen disciplines: Genetics
                Data availability:
                ScienceOpen disciplines: Genetics

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