RESUMO
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.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteômica/métodos , Interferência de RNA , Precursores de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , TransfecçãoRESUMO
Cajal bodies (CBs) are evolutionarily conserved nuclear structures involved in the metabolism of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). CBs are not present in all cell types, and the trigger for their formation is not yet known. Here, we depleted cells of factors required for the final steps of snRNP assembly and assayed for the presence of stalled intermediates in CBs. We show that depletion induces formation of CBs in cells that normally lack these nuclear compartments, suggesting that CB nucleation is triggered by an imbalance in snRNP assembly. Accumulation of stalled intermediates in CBs depends on the di-snRNP assembly factor SART3. SART3 is required for both the induction of CB formation as well as the tethering of incomplete snRNPs to coilin, the CB scaffolding protein. We propose a model wherein SART3 monitors tri-snRNP assembly and sequesters incomplete particles in CBs, thereby allowing cells to maintain a homeostatic balance of mature snRNPs in the nucleoplasm.
