A multi-factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing

Alexandra Bergfort,Gert Weber,Benno Kuropka,Tarek Hilal,İbrahim Avşar Ilik,Florian Heyd,Markus C Wahl,Christian Freund,Tuğçe Aktaş,Marco Preußner

doi:10.1038/s41467-022-28754-2

Abstract

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identify additional C9ORF78 interactors in spliceosomes. Cryogenic electron microscopy structures reveal how C9ORF78 and the spliceosomal B complex protein, FBP21, wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock-down of C9ORF78 leads to alternative NAGNAG 3′-splice site usage and exon skipping, the latter dependent on BRR2. Inspection of spliceosome structures shows that C9ORF78 could contact several detected spliceosome interactors when bound to BRR2, including the suggested 3′-splice site regulating helicase, PRPF22. Together, our data establish C9ORF78 as a late-stage splicing regulatory protein that takes advantage of a multi-factor trafficking site on BRR2, providing one explanation for suggested roles of BRR2 during splicing catalysis and alternative splicing.

Highlights

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear
BRR2 is a specific subunit of the U5 small nuclear RNP that plays a pivotal role during spliceosome activation, the step during which the most substantial rearrangements occur[7,8]
GSTC9ORF78 stably interacted with full-length BRR2 (BRR2FL) in analytical size exclusion chromatography (SEC; Fig. 1a)

Summary

Introduction

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. BRR2 is a specific subunit of the U5 small nuclear (sn) RNP that plays a pivotal role during spliceosome activation, the step during which the most substantial rearrangements occur[7,8] During this transition, BRR2 unwinds the initially base-paired U4/U6 di-snRNA, facilitating the release of U4 snRNA and of all U4/U6 di-snRNP-associated proteins, and allowing U6 to form a catalytically important stem-loop and to engage in alternative interactions with the 5′-splice site (ss) and U2 snRNA7–9. BRR2 has been suggested to be involved in the catalytic and disassembly phases of splicing, but the underlying mechanisms are not understood, as these stages do not present known RNA/RNP substrates on which the remodeling factor might act, and as BRR2’s ATPase/helicase activity does not seem to be required during these stages[10–12].

Methods

Results

Conclusion