Structural Basis of Branch Site Recognition by the Human Spliceosome

Overview

Journal Science

Specialty Science

Date 2021 Nov 25

PMID 34822310

Citations 28

Authors

Jonas Tholen

Michal Razew

Felix Weis

Wojciech P Galej

Affiliations

Soon will be listed here.

Abstract

Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human 17 U2 snRNP and reconstituted in vitro its adenosine 5´-triphosphate (ATP)–dependent remodeling and binding to the pre–messenger RNA substrate. We determined a series of high-resolution (2.0 to 2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2 snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism based on branch helix stability.

Citing Articles

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References

Kao C, Cao E, Wai H, Cheng S . Evidence for complex dynamics during U2 snRNP selection of the intron branchpoint. Nucleic Acids Res. 2021; 49(17):9965-9977. PMC: 8464032. DOI: 10.1093/nar/gkab695. View

Smith D, Konarska M, Query C . Insights into branch nucleophile positioning and activation from an orthogonal pre-mRNA splicing system in yeast. Mol Cell. 2009; 34(3):333-43. PMC: 2730498. DOI: 10.1016/j.molcel.2009.03.012. View

Wu J, Manley J . Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989; 3(10):1553-61. DOI: 10.1101/gad.3.10.1553. View

Pettersen E, Goddard T, Huang C, Meng E, Couch G, Croll T . UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 2020; 30(1):70-82. PMC: 7737788. DOI: 10.1002/pro.3943. View

Liang W, Cheng S . A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence. Genes Dev. 2015; 29(1):81-93. PMC: 4281567. DOI: 10.1101/gad.253708.114. View

Franken H, Mathieson T, Childs D, Sweetman G, Werner T, Togel I . Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry. Nat Protoc. 2015; 10(10):1567-93. DOI: 10.1038/nprot.2015.101. View

Williams C, Headd J, Moriarty N, Prisant M, Videau L, Deis L . MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. 2017; 27(1):293-315. PMC: 5734394. DOI: 10.1002/pro.3330. View

Michaud S, REED R . An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 1991; 5(12B):2534-46. DOI: 10.1101/gad.5.12b.2534. View

Newnham C, Query C . The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5' to the branch site. RNA. 2001; 7(9):1298-309. PMC: 1370173. DOI: 10.1017/s1355838201010561. View

10.

Kastner B, Fischer N, Golas M, Sander B, Dube P, Boehringer D . GraFix: sample preparation for single-particle electron cryomicroscopy. Nat Methods. 2007; 5(1):53-5. DOI: 10.1038/nmeth1139. View

11.

Schellenberg M, Edwards R, Ritchie D, Kent O, Golas M, Stark H . Crystal structure of a core spliceosomal protein interface. Proc Natl Acad Sci U S A. 2006; 103(5):1266-71. PMC: 1360545. DOI: 10.1073/pnas.0508048103. View

12.

Plaschka C, Lin P, Nagai K . Structure of a pre-catalytic spliceosome. Nature. 2017; 546(7660):617-621. PMC: 5503131. DOI: 10.1038/nature22799. View

13.

Murshudov G, Skubak P, Lebedev A, Pannu N, Steiner R, Nicholls R . REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011; 67(Pt 4):355-67. PMC: 3069751. DOI: 10.1107/S0907444911001314. View

14.

Query C, McCaw P, Sharp P . A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract. Mol Cell Biol. 1997; 17(5):2944-53. PMC: 232146. DOI: 10.1128/MCB.17.5.2944. View

15.

MacMillan A, Query C, Allerson C, Chen S, Verdine G, Sharp P . Dynamic association of proteins with the pre-mRNA branch region. Genes Dev. 1994; 8(24):3008-20. DOI: 10.1101/gad.8.24.3008. View

16.

Cretu C, Gee P, Liu X, Agrawal A, Nguyen T, Ghosh A . Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors. Nat Commun. 2021; 12(1):4491. PMC: 8302644. DOI: 10.1038/s41467-021-24741-1. View

17.

Cretu C, Agrawal A, Cook A, Will C, Fekkes P, Smith P . Structural Basis of Splicing Modulation by Antitumor Macrolide Compounds. Mol Cell. 2018; 70(2):265-273.e8. DOI: 10.1016/j.molcel.2018.03.011. View

18.

Rohou A, Grigorieff N . CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J Struct Biol. 2015; 192(2):216-21. PMC: 6760662. DOI: 10.1016/j.jsb.2015.08.008. View

19.

Punjani A, Rubinstein J, Fleet D, Brubaker M . cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods. 2017; 14(3):290-296. DOI: 10.1038/nmeth.4169. View

20.

Haselbach D, Komarov I, Agafonov D, Hartmuth K, Graf B, Dybkov O . Structure and Conformational Dynamics of the Human Spliceosomal B Complex. Cell. 2018; 172(3):454-464.e11. DOI: 10.1016/j.cell.2018.01.010. View