Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones.
ABSTRACT: 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.
Project description:U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo an intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before a next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a new component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.
Project description:Mutations in genes associated with the U4/U6-U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome are implicated in autosomal-dominant retinitis pigmentosa (adRP), a group of progressive retinal degenerative disorders leading to visual impairment, loss of visual field, and even blindness. We recently assigned a locus (RP33) for adRP to 2cen-q12.1, a region that harbors the SNRNP200 gene encoding hBrr2, another U4/U6-U5 snRNP component that is required for unwinding of U4/U6 snRNAs during spliceosome activation and for disassembly of the spliceosome. Here, we report the identification of a missense mutation, c.3260C>T (p.S1087L), in exon 25 of the SNRNP200 gene in an RP33-linked family. The c.3260C>T substitution showed complete cosegregation with the retinitis pigmentosa (RP) phenotype over four generations, but was absent in a panel of 400 controls. The p.S1087L mutation and p.R1090L, another adRP-associated allele, reside in the "ratchet" helix of the first of two Sec63 domains implicated in the directionality and processivity of nucleic acid unwinding. Indeed, marked defects in U4/U6 unwinding, but not U4/U6-U5 snRNP assembly, were observed in budding yeast for the analogous mutations (N1104L and R1107L) of the corresponding Brr2p residues. The linkage of hBrr2 to adRP suggests that the mechanism of pathogenesis for splicing-factor-related RP may fundamentally derive from a defect in hBrr2-dependent RNA unwinding and a consequent defect in spliceosome activation.
Project description:Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina.
Project description:Aged and damaged mitochondria can be selectively degraded by specific autophagic elimination, termed mitophagy. Defects in mitophagy have been increasingly linked to several diseases including neurodegenerative diseases, metabolic diseases and other aging-related diseases. However, the molecular mechanisms of mitophagy are not fully understood. Here, we identify PRPF8 (pre-mRNA processing factor 8), a core component of the spliceosome, as an essential mediator in hypoxia-induced mitophagy from an RNAi screen based on a fluorescent mitophagy reporter, mt-Keima. Knockdown of PRPF8 significantly impairs mitophagosome formation and subsequent mitochondrial clearance through the aberrant mRNA splicing of ULK1, which mediates macroautophagy/autophagy initiation. Importantly, autosomal dominant retinitis pigmentosa (adRP)-associated PRPF8 mutant R2310K is defective in regulating mitophagy. Moreover, knockdown of other adRP-associated splicing factors, including PRPF6, PRPF31 and SNRNP200, also lead to ULK1 mRNA mis-splicing and mitophagy defects. Thus, these findings demonstrate that PRPF8 is essential for mitophagy and suggest that dysregulation of spliceosome-mediated mitophagy may contribute to pathogenesis of retinitis pigmentosa.
Project description:RNA splicing is a fundamental process for protein synthesis. Recent studies have reported that drugs that inhibit splicing have cytotoxic effects on various tumor cell lines. In this report, we demonstrate that depletion of SNW1, a component of the spliceosome, induces apoptosis in breast cancer cells. Proteomics and biochemical analyses revealed that SNW1 directly associates with other spliceosome components, including EFTUD2 (Snu114) and SNRNP200 (Brr2). The SKIP region of SNW1 interacted with the N-terminus of EFTUD2 as well as two independent regions in the C-terminus of SNRNP200. Similar to SNW1 depletion, knockdown of EFTUD2 increased the numbers of apoptotic cells. Furthermore, we demonstrate that exogenous expression of either the SKIP region of SNW1 or the N-terminus region of EFTUD2 significantly promoted cellular apoptosis. Our results suggest that the inhibition of SNW1 or its associating proteins may be a novel therapeutic strategy for cancer treatment.
Project description:The U5 small nuclear ribonucleoprotein particle (snRNP) forms the heart of the spliceosome which is required for intron removal from pre-mRNA. The proteins Prp8, Snu114 and Brr2 all assemble with the U5 small nuclear RNA (snRNA) to produce the U5 snRNP. Successful assembly of the U5 snRNP, then incorporation of this snRNP into the U4/U6.U5 tri-snRNP and the spliceosome, is essential for producing an active spliceosome. We have investigated the requirements for Prp8, Snu114 and Brr2 association with the U5 snRNA to form the U5 snRNP in yeast. Mutations were constructed in the highly conserved loop 1 and internal loop 1 (IL1) of the U5 snRNA and their function assessed in vivo. The influence of these U5 mutations on association of Prp8, Snu114 and Brr2 with the U5 snRNA were then determined. U5 snRNA loop 1 and both sides of IL1 in U5 were important for association of Prp8, Snu114 and Brr2 with the U5 snRNA. Mutations in the 3' side of U5 IL1 resulted in the greatest reduction of Prp8, Snu114 and Brr2 association with the U5 snRNA. Genetic screening of brr2 and U5 snRNA mutants revealed synthetic lethal interactions between alleles in Brr2 and the 3' side of U5 snRNA IL1 which reflects reduced association between Brr2 and U5 IL1. We propose that the U5 snRNA IL1 is a platform for protein binding and is required for Prp8, Brr2 and Snu114 association with the U5 snRNA to form the U5 snRNP.
Project description:The stepwise assembly of the highly dynamic spliceosome is guided by RNA-dependent ATPases of the DEAD-box family, whose regulation is poorly understood. In the canonical assembly model, the U4/U6.U5 triple snRNP binds only after joining of the U1 and, subsequently, U2 snRNPs to the intron-containing pre-mRNA. Catalytic activation requires the exchange of U6 for U1 snRNA at the 5' splice site, which is promoted by the DEAD-box protein Prp28. Because Prp8, an integral U5 snRNP protein, is thought to be a central regulator of DEAD-box proteins, we conducted a targeted search in Prp8 for cold-insensitive suppressors of a cold-sensitive Prp28 mutant, prp28-1. We identified a cluster of suppressor mutations in an N-terminal bromodomain-like sequence of Prp8. To identify the precise defect in prp28-1 strains that is suppressed by the Prp8 alleles, we analyzed spliceosome assembly in vivo and in vitro. Surprisingly, in the prp28-1 strain, we observed a block not only to spliceosome activation but also to one of the earliest steps of assembly, formation of the ATP-independent commitment complex 2 (CC2). The Prp8 suppressor partially corrected both the early assembly and later activation defects of prp28-1, supporting a role for this U5 snRNP protein in both the ATP-independent and ATP-dependent functions of Prp28. We conclude that the U5 snRNP has a role in the earliest events of assembly, prior to its stable incorporation into the spliceosome.
Project description:We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.
Project description:The pre-catalytic spliceosome (B complex) is preceded by its precursor spliceosome (pre-B complex) and followed by the activated spliceosome (Bact complex). The pre-B-to-B and B-to-Bact transitions are driven by the ATPase/helicases Prp28 and Brr2, respectively. In this study, we report the cryo-electron microscopy structures of the human pre-B complex and the human B complex at an average resolution of 5.7 and 3.8 Å, respectively. In the pre-B complex, U1 and U2 small nuclear ribonucleoproteins (snRNPs) associate with two edges of the tetrahedron-shaped U4/U6.U5 tri-snRNP. The pre-mRNA is yet to be recognized by U5 or U6 small nuclear RNA (snRNA), and loop I of U5 snRNA remains unengaged. In the B complex, U1 snRNP and Prp28 are dissociated, the 5'-exon is anchored to loop I of U5 snRNA, and the 5'-splice site is recognized by U6 snRNA through duplex formation. In sharp contrast to S. cerevisiae, most components of U2 snRNP and tri-snRNP, exemplified by Brr2, undergo pronounced rearrangements in the human pre-B-to-B transition. Structural analysis reveals mechanistic insights into the assembly and activation of the human spliceosome.
Project description:Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.