JCB_201701165 1579..1596

Pre-mRNA splicing is a crucial step in eukaryotic gene expression that greatly diversifies the proteome. Splicing is catalyzed by a complex called the spliceosome, which consists of five major small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins. Each snRNP contains a short noncoding uridine-rich RNA (snRNA), a ring of seven Sm or Like-Sm (in the case of U6 RNP) proteins, and 1–12 proteins that are specific for each snRNP (Matera and Wang, 2014). The assembly of Sm-type snRNPs is a complex process that involves both cytoplasmic and nuclear phases. In metazoans, the nascent snRNAs are rapidly exported to the cytoplasm, where they associate with the survival of motor neurons (SMN) complex. This promotes the assembly of the Sm ring around a specific snRNA sequence (Matera and Wang, 2014; Raimer et al., 2016), and this core snRNP is then reimported to the nucleus, where it first appears in nuclear structures called Cajal bodies (CBs; Sleeman and Lamond, 1999). Here, the final stages of snRNP biogenesis occur, namely snRNA modifications and formation of mature snRNPs, which include the addition of snRNP-specific proteins and assembly of complex snRNPs (Staněk, 2016). Although the assembly of the Sm core by the SMN complex has been studied in great detail, little is known about the biogenesis of snRNP-specific proteins despite their importance for splicing. Recent structural studies show that the U5 snRNP plays a central role in the spliceosome and particularly the highly conserved U5 protein PRPF8 (Yan et al., 2015; Bertram et al., 2017). PRPF8 adopts a complex 3D structure with a central cavity that forms the catalytic center of the spliceosome. PRPF8 also holds two other U5-specific proteins that perform essential functions during splicing: the helicase SNR NP200 (Brr2 in yeast) and the GTPase EFT UD2 (Snu114 in yeast). SNR NP200 unwinds the U4/U6 duplex to activate the spliceosome, whereas EFT UD2, along with PRPF8, controls SNR NP200 activity (Kuhn et al., 1999; Small et al., 2006; Maeder et al., 2009; 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 EFT UD2 and SNR NP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFT UD2, and SNR NP200 in association with the HSP90/R2TP complex, its ZNH IT2 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. Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones