The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling

Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4’s intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling

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

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

Structural and functional analysis of BRR2 interactors C9ORF78 and TSSC4 and their role in pre mRNA splicing

Pre mRNA splicing is a crucial step in eukaryotic gene expression, in which non coding introns are excised and coding exons are ligated. Alternative splicing, a mechanism by which different combinations of exons are joined, dramatically increases proteomic complexity and aberrations in this process are often linked to human disease. The splicing reactions are carried out by the spliceosome, a dynamic ribonucleoprotein machinery that assembles stepwise on the pre mRNA and adopts at least 10 distinct conformational states. Eight conserved RNA helicases drive and control the conformational and compositional rearrangements of the spliceosome, one of which is BRR2. BRR2 enters the spliceosome as part of the tri snRNP and stays associated during all remaining phases of splicing. Hence, its function needs to be tightly regulated. Apart from the PRP8 JAB1 domain, which binds the BRR2 N terminal helicase cassette and regulates its activity, the intrinsically disordered protein IDP FBP21 has recently been found to bind the BRR2 C terminal helicase cassette and exert an inhibiting function. In this study, the interaction of human BRR2 with two further IDPs, C9ORF78 and TSSC4, was validated in vitro and in human cells. The binding interface was studied by protein interaction assays and cryo EM structures were obtained for the BRR2 helicase region in complex with C9ORF78, TSSC4 and FBP21, respectively. Competitive binding of the three BRR2 interactors was investigated and for TSSC4 and C9ORF78 the nuclear interactomes were analyzed by FLAG IP followed by proteomics. Furthermore, the C9ORF78 and TSSC4 functions in splicing were studied via a siRNA mediated knockdown combined with RNA sequencing. The data presented here demonstrate mutually exclusive binding of the BRR2 C cassette by TSSC4, C9ORF78 and FBP21. The proteomics results implicate functions of C9ORF78 and TSSC4 in several nuclear pathways, including splicing. Both proteins were identified as splicing factors that regulate alternative splicing of specific target pre mRNAs, mainly via intronic motifs. In case of C9ORF78, the data suggest a model in which C9ORF78 promotes exon inclusion in specific target mRNAs by facilitating BRR2 mediated unwinding of secondary structure forming introns. While this model will require further validation, the findings in this work point towards an unprecedented function of BRR2 in alternative splicin