Journal: Nucleic Acids Res / Year: 2022 Title: The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling. Authors: Alexandra Bergfort / Tarek Hilal / Benno Kuropka / İbrahim Avşar Ilik / Gert Weber / Tuğçe Aktaş / Christian Freund / Markus C Wahl / Abstract: 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. ...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.
History
Deposition
Oct 7, 2021
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Header (metadata) release
Jan 26, 2022
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Map release
Jan 26, 2022
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Update
Aug 10, 2022
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Current status
Aug 10, 2022
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3349 / Average exposure time: 30.59 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Particle selection
Number selected: 1025529
CTF correction
Software - Name: cryoSPARC (ver. 2.9)
Initial angle assignment
Type: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.9)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9)
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 387973
FSC plot (resolution estimation)
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