Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2018 Dec 4

PMID 30503210

Citations 66

Authors

Lilan You

Jun Ma

Jiuyu Wang

Daria Artamonova

Min Wang

Liang Liu

Hua Xiang

Konstantin Severinov

Xinzheng Zhang

Yanli Wang

Affiliations

Soon will be listed here.

Abstract

Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.

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References

Kimanius D, Forsberg B, Scheres S, Lindahl E . Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Elife. 2016; 5. PMC: 5310839. DOI: 10.7554/eLife.18722. View

Taylor D, Zhu Y, Staals R, Kornfeld J, Shinkai A, van der Oost J . Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning. Science. 2015; 348(6234):581-5. PMC: 4582657. DOI: 10.1126/science.aaa4535. View

Niewoehner O, Jinek M . Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6. RNA. 2016; 22(3):318-29. PMC: 4748810. DOI: 10.1261/rna.054098.115. View

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S . CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315(5819):1709-12. DOI: 10.1126/science.1138140. View

Ramia N, Spilman M, Tang L, Shao Y, Elmore J, Hale C . Essential structural and functional roles of the Cmr4 subunit in RNA cleavage by the Cmr CRISPR-Cas complex. Cell Rep. 2014; 9(5):1610-1617. PMC: 4269474. DOI: 10.1016/j.celrep.2014.11.007. View

Henne A, Bruggemann H, Raasch C, Wiezer A, Hartsch T, Liesegang H . The genome sequence of the extreme thermophile Thermus thermophilus. Nat Biotechnol. 2004; 22(5):547-53. DOI: 10.1038/nbt956. View

Emsley P, Lohkamp B, Scott W, Cowtan K . Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 4):486-501. PMC: 2852313. DOI: 10.1107/S0907444910007493. View

Niewoehner O, Garcia-Doval C, Rostol J, Berk C, Schwede F, Bigler L . Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature. 2017; 548(7669):543-548. DOI: 10.1038/nature23467. View

Osawa T, Inanaga H, Sato C, Numata T . Crystal structure of the CRISPR-Cas RNA silencing Cmr complex bound to a target analog. Mol Cell. 2015; 58(3):418-30. DOI: 10.1016/j.molcel.2015.03.018. View

10.

Makarova K, Wolf Y, Alkhnbashi O, Costa F, Shah S, Saunders S . An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015; 13(11):722-36. PMC: 5426118. DOI: 10.1038/nrmicro3569. View

11.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N . The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16):2078-9. PMC: 2723002. DOI: 10.1093/bioinformatics/btp352. View

12.

Hale C, Zhao P, Olson S, Duff M, Graveley B, Wells L . RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell. 2009; 139(5):945-56. PMC: 2951265. DOI: 10.1016/j.cell.2009.07.040. View

13.

Samai P, Pyenson N, Jiang W, Goldberg G, Hatoum-Aslan A, Marraffini L . Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity. Cell. 2015; 161(5):1164-1174. PMC: 4594840. DOI: 10.1016/j.cell.2015.04.027. View

14.

Spilman M, Cocozaki A, Hale C, Shao Y, Ramia N, Terns R . Structure of an RNA silencing complex of the CRISPR-Cas immune system. Mol Cell. 2013; 52(1):146-52. PMC: 3864027. DOI: 10.1016/j.molcel.2013.09.008. View

15.

Rouillon C, Zhou M, Zhang J, Politis A, Beilsten-Edmands V, Cannone G . Structure of the CRISPR interference complex CSM reveals key similarities with cascade. Mol Cell. 2013; 52(1):124-34. PMC: 3807668. DOI: 10.1016/j.molcel.2013.08.020. View

16.

Elmore J, Sheppard N, Ramia N, Deighan T, Li H, Terns R . Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system. Genes Dev. 2016; 30(4):447-59. PMC: 4762429. DOI: 10.1101/gad.272153.115. View

17.

Goldberg G, Jiang W, Bikard D, Marraffini L . Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting. Nature. 2014; 514(7524):633-7. PMC: 4214910. DOI: 10.1038/nature13637. View

18.

de Grado M, Castan P, Berenguer J . A high-transformation-efficiency cloning vector for Thermus thermophilus. Plasmid. 1999; 42(3):241-5. DOI: 10.1006/plas.1999.1427. View

19.

Sheppard N, Glover 3rd C, Terns R, Terns M . The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease. RNA. 2015; 22(2):216-24. PMC: 4712672. DOI: 10.1261/rna.039842.113. View

20.

Tamulaitis G, Kazlauskiene M, Manakova E, Venclovas C, Nwokeoji A, Dickman M . Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus. Mol Cell. 2014; 56(4):506-17. DOI: 10.1016/j.molcel.2014.09.027. View