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

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

Nucleic acids research 2022 Mar 21

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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. © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

Citation

Alexandra Bergfort, Tarek Hilal, Benno Kuropka, İbrahim Avşar Ilik, Gert Weber, Tuğçe Aktaş, Christian Freund, Markus C Wahl. The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling. Nucleic acids research. 2022 Mar 21;50(5):2938-2958