Splicing is catalyzed by the spliceosome, a complex of five major small nuclear ribonucleoprotein particles (snRNPs). The pre-mRNA splicing factor PRPF8 is a crucial component of the U5 snRNP, and together with EFTUD2 and SNRNP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFTUD2, and SNRNP200 in association with the HSP90/R2TP complex, its ZNHIT2 cofactor, and additional proteins. HSP90 and R2TP bind unassembled U5 proteins in the cytoplasm, stabilize them, and promote the formation of the U5 snRNP. We further found that PRPF8 mutants causing Retinitis pigmentosa assemble less efficiently with the U5 snRNP and bind more strongly to R2TP, with one mutant retained in the cytoplasm in an R2TP-dependent manner. We propose that the HSP90/R2TP chaperone system promotes the assembly of a key module of U5 snRNP while assuring the quality control of PRPF8. The proteomics data further reveal new interactions between R2TP and the tuberous sclerosis complex (TSC), pointing to a potential link between growth signals and the assembly of key cellular machines.

Faculty of Science Charles University Prague 128 00 Prague Czech Republic

Institut de Génétique Moléculaire de Montpellier Centre National de la Recherche Scientifique University of Montpellier 34293 Montpellier France

Institut de Génomique Fonctionnelle Centre National de la Recherche Scientifique University of Montpellier 34090 Montpellier France

Institute of Molecular Genetics Czech Academy of Sciences 142 20 Prague Czech Republic

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Retinitis pigmentosa-linked mutations impair the snRNA unwinding activity of SNRNP200 and reduce pre-mRNA binding of PRPF8

Coilin and Cajal bodies

Retinitis pigmentosa-associated mutations in mouse Prpf8 cause misexpression of circRNAs and degeneration of cerebellar granule cells

Retinitis pigmentosa-linked mutation in DHX38 modulates its splicing activity

TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation

DIS3L2 and LSm proteins are involved in the surveillance of Sm ring-deficient snRNAs

The Sm-core mediates the retention of partially-assembled spliceosomal snRNPs in Cajal bodies until their full maturation

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PDB
4KIT