psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

Lisa M. Strittmatter, Charlotte Capitanchik, Andrew J. Newman, Martina Hallegger, Christine M. Norman, Sebastian M. Fica, Chris Oubridge, Nicholas M. Luscombe, Jernej Ule () and Kiyoshi Nagai
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Lisa M. Strittmatter: MRC Laboratory of Molecular Biology
Charlotte Capitanchik: The Francis Crick Institute
Andrew J. Newman: MRC Laboratory of Molecular Biology
Martina Hallegger: The Francis Crick Institute
Christine M. Norman: MRC Laboratory of Molecular Biology
Sebastian M. Fica: MRC Laboratory of Molecular Biology
Chris Oubridge: MRC Laboratory of Molecular Biology
Nicholas M. Luscombe: The Francis Crick Institute
Jernej Ule: The Francis Crick Institute
Kiyoshi Nagai: MRC Laboratory of Molecular Biology

Nature Communications, 2021, vol. 12, issue 1, 1-15

Abstract: Abstract RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3′-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.

Date: 2021
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DOI: 10.1038/s41467-021-21745-9

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