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The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing 1 . Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP5 2-7 . Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL) 8 , but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers 9 , contains a HEAT domain (SF3B1 HEAT ) with an open conformation in isolated SF3b 10 , but a closed conformation in spliceosomes 11 , which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1 HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1 HEAT . Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.

Titel:	Molecular architecture of the human 17S U2 snRNP.
Autor/in / Beteiligte Person:	Zhang, Z ; Will, CL ; Bertram, K ; Dybkov, O ; Hartmuth, K ; Agafonov, DE ; Hofele, R ; Urlaub, H ; Kastner, B ; Lührmann, R ; Stark, H
Zeitschrift:	Nature, Jg. 583 (2020-07-01), Heft 7815, S. 310-313
Veröffentlichung:	Basingstoke : Nature Publishing Group ; <i>Original Publication</i>: London, Macmillan Journals ltd., 2020
Medientyp:	academicJournal
ISSN:	1476-4687 (electronic)
DOI:	10.1038/s41586-020-2344-3
Schlagwort:	Base Sequence DEAD-box RNA Helicases chemistry DEAD-box RNA Helicases metabolism HeLa Cells Humans Models, Molecular Phosphoproteins chemistry Phosphoproteins metabolism Protein Binding Protein Conformation RNA Splicing Factors chemistry RNA Splicing Factors metabolism Ribonucleoprotein, U2 Small Nuclear genetics Ribonucleoprotein, U2 Small Nuclear metabolism Trans-Activators chemistry Trans-Activators metabolism Cryoelectron Microscopy Ribonucleoprotein, U2 Small Nuclear chemistry Ribonucleoprotein, U2 Small Nuclear ultrastructure
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Research Support, Non-U.S. Gov't Language: English [Nature] 2020 Jul; Vol. 583 (7815), pp. 310-313. <i>Date of Electronic Publication: </i>2020 Jun 03. MeSH Terms: Cryoelectron Microscopy* ; Ribonucleoprotein, U2 Small Nuclear / chemistry ; Ribonucleoprotein, U2 Small Nuclear / ultrastructure ; Base Sequence ; DEAD-box RNA Helicases / chemistry ; DEAD-box RNA Helicases / metabolism ; HeLa Cells ; Humans ; Models, Molecular ; Phosphoproteins / chemistry ; Phosphoproteins / metabolism ; Protein Binding ; Protein Conformation ; RNA Splicing Factors / chemistry ; RNA Splicing Factors / metabolism ; Ribonucleoprotein, U2 Small Nuclear / genetics ; Ribonucleoprotein, U2 Small Nuclear / metabolism ; Trans-Activators / chemistry ; Trans-Activators / metabolism References: Will, C. L. & Lührmann, R. Spliceosome structure and function. Cold Spring Harb. Perspect. Biol. 3, a003707 (2011). 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Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat. Commun. 7, 10615 (2016). (PMID: 26842708474300910.1038/ncomms10615) ; Zhang, J. et al. Disease-causing mutations in SF3B1 alter splicing by disrupting interaction with SUGP1. Mol. Cell 76, 82–95.e7 (2019). (PMID: 31474574706527310.1016/j.molcel.2019.07.017) ; Kielkopf, C. L., Lücke, S. & Green, M. R. U2AF homology motifs: protein recognition in the RRM world. Genes Dev. 18, 1513–1526 (2004). (PMID: 1523173310.1101/gad.1206204) Substance Nomenclature: 0 (HTATSF1 protein, human) ; 0 (Phosphoproteins) ; 0 (RNA Splicing Factors) ; 0 (Ribonucleoprotein, U2 Small Nuclear) ; 0 (SF3B1 protein, human) ; 0 (Trans-Activators) ; EC 3.6.1.- (DDX46 protein, human) ; EC 3.6.4.13 (DEAD-box RNA Helicases) Entry Date(s): Date Created: 20200605 Date Completed: 20200810 Latest Revision: 20220418 Update Code: 20240513

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Molecular architecture of the human 17S U2 snRNP.