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

Lisa M Strittmatter,Nicholas M Luscombe,Chris Oubridge,Jernej Ule,Martina Hallegger,Kiyoshi Nagai,Christine M Norman,Charlotte Capitanchik,Andrew J Newman,Sebastian M Fica

doi:10.1038/s41467-021-21745-9

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.

Highlights

RNA helicases remodel the spliceosome to enable pre-mature RNA (mRNA) splicing, but their binding and mechanism of action remain poorly understood
Optimized purified spliceosome iCLIP (psiCLIP) detects RNA binding with positional specificity
As in conventional iCLIP, spliceosomes are irradiated with 254 nm ultraviolet light to crosslink proteins to the pre-mRNA substrate and snRNAs

Summary

Introduction

RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. Prp[16] repositions the substrate after the branching reaction by removing the branch helix, formed between the branch point sequence and U2 snRNA, from the active site of the C-complex spliceosome[8,9,10]. Following this remodelling event, Prp[16] dissociates from the C complex and causes dissociation of the branching factors that stabilize the branch helix[11]. Exon ligation results in the formation of the spliceosomal P complex that retains both the excised intron and the mature mRNA, which is held by spliceosomal proteins and the U5 snRNA11. It remained unclear whether Prp[22] translocates directly through the duplex or acts at a distance

Methods

Results

Conclusion