Nuclear Ribonucleoprotein Particles Research Articles

RETRACTED: circTulp4 functions in Alzheimer’s disease pathogenesis by regulating its parental gene, Tulp4

Alzheimer's disease (AD)-one of the most common neurodegenerative diseases worldwide-impairs cognition, memory, and language ability and causes dementia. However, AD pathogenesis remains poorly elucidated. Recently, a potential link between AD and circular RNAs (circRNAs) has been uncovered, but only a few circRNAs that might be involved in AD have been identified. Here, we systematically investigated circRNAs in the APP/PS1 model mouse brain through deep RNA sequencing. We report that circRNAs are markedly enriched in the brain and that several circRNAs exhibit differential expression between wild-type and APP/PS1 mice. We characterized one abundant circRNA, circTulp4, derived from Intron1 of the gene Tulp4. Our results indicate that circTulp4 predominantly localizes in the nucleus and interacts with U1 small nuclear ribonucleoprotein particle (snRNP) and RNA polymerase II to modulate the transcription of its parental gene, Tulp4, and thereby regulate the function of the nervous system, and might participate in the development of AD.

Ecd promotes U5 snRNP maturation and Prp8 stability.

Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs), the biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches, we demonstrate the Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. We show that the predominant task of Ecd is to deliver Prp8 to the emerging U5 snRNPs in the cytoplasm. Ecd deficiency, on the other hand, leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on our findings, we propose that Ecd chaperones Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis pathway necessary to meet the cellular demand for functional spliceosomes.

A tight couple makes messenger RNAs

RNA Splicing Gene expression in eukaryotes first requires transcription of DNA to an RNA copy and then splicing to form the final, processed messenger RNA (mRNA). Zhang et al. investigated how gene transcription and RNA splicing are physically coupled. Using cryo–electron microscopy, they resolved the molecular structure of a complex of the transcription enzyme RNA polymerase II with part of the splicing machinery, the U1 small nuclear ribonucleoprotein particle. The results provide important details for our understanding of coupled mRNA production. Science , this issue p. [305][1] [1]: /lookup/doi/10.1126/science.abf1870

Structure of a transcribing RNA polymerase II-U1 snRNP complex.

To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo-electron microscopy structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5' splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5' splice site, leading to the formation of a "growing intron loop." Loop formation may facilitate scanning of nascent pre-mRNA for the 3' splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative splicing.

A study on allosteric communication in U1A-snRNA binding interactions: network analysis combined with molecular dynamics data

The allosteric regulation during the binding interactions between small nuclear RNAs (snRNAs) and the associated protein factors is critical to the function of spliceosomes in alternative RNA splicing. Although network models combined with molecular dynamics simulations have shown to be powerful tools for the analysis of protein allostery, the atomic-level simulations are, however, too expensive and with limited accuracy for the large-size systems. In this work, we use a residual network model combined with a coarse-grained Gaussian network model (GNM) to investigate the binding interactions between the snRNA and the human U1A protein which is a major component of the spliceosomal U1 small nuclear ribonucleoprotein particle, and to identify the residues that play an important role in the allosteric communication in U1A during this process. We also utilize the Girvan-Newman method to detect the structural organization in U1A-snRNA recognition and interactions. Our results reveal that: (Ι) not only the residues at the binding sites that are traditionally considered to play a major role in U1A-snRNA association, but those residues that are far away from the RNA binding interface participate in the U1A's allosteric signal transmission induced by the RNA binding; (Π) the structure of U1A protein is well organized with different communities acting different roles for its RNA binding and allosteric regulation. The study demonstrates that the combination of the residual network and elastic network models is an effective and efficient method which can be readily extended to the investigation of the allosteric communication for other macromolecular interaction systems.

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

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo a complex maturation pathway containing multiple steps in the nucleus and in the cytoplasm. snRNP biogenesis is strictly proofread and several quality control checkpoints are placed along the pathway. Here, we analyzed the fate of small nuclear RNAs (snRNAs) that are unable to acquire a ring of Sm proteins. We showed that snRNAs lacking the Sm ring are unstable and accumulate in P-bodies in an LSm1-dependent manner. We further provide evidence that defective snRNAs without the Sm binding site are uridylated at the 3′ end and associate with DIS3L2 3′→5′ exoribonuclease and LSm proteins. Finally, inhibition of 5′→3′ exoribonuclease XRN1 increases association of ΔSm snRNAs with DIS3L2, which indicates competition and compensation between these two degradation enzymes. Together, we provide evidence that defective snRNAs without the Sm ring are uridylated and degraded by alternative pathways involving either DIS3L2 or LSm proteins and XRN1.

Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA.

Deregulation of pre-mRNA splicing is observed in many cancers and hematological malignancies. Genes encoding splicing factors are frequently mutated in myelodysplastic syndromes, in which SF3B1 mutations are the most frequent. SF3B1 is an essential component of the U2 small nuclear ribonucleoprotein particle that interacts with branch point sequences close to the 3’ splice site during pre-mRNA splicing. SF3B1 mutations mostly lead to substitutions at restricted sites in the highly conserved HEAT domain, causing a modification of its function. We found that SF3B1 was aberrantly spliced in various neoplasms carrying an SF3B1 mutation, by exploring publicly available RNA sequencing raw data. We aimed to characterize this novel SF3B1 transcript, which is expected to encode a protein with an insertion of eight amino acids in the H3 repeat of the HEAT domain. We investigated the splicing proficiency of this SF3B1 protein isoform, in association with the most frequent mutation (K700E), through functional complementation assays in two myeloid cell lines stably expressing distinct SF3B1 variants. The yeast Schizosaccharomyces pombe was also used as an alternative model. Insertion of these eight amino acids in wild-type or mutant SF3B1 (K700E) abolished SF3B1 essential function, highlighting the crucial role of the H3 repeat in the splicing function of SF3B1.

Phenotypic analysis combined with tandem mass tags (TMT) labeling reveal the heterogeneity of strawberry stolon buds

BackgroundRamet propagation in strawberry (Fragaria × ananassa) is the most effective way in production. However, the lack of systematically phenotypic observations and high-throughput methods limits our ability to analyze the key factors regulating the heterogeneity in strawberry stolon buds.ResultsFrom observation, we found that the axillary bud located in the first node quickly stepped into dormancy (DSB), after several bract and leaf buds were differentiated. The stolon apical meristem (SAM) degenerated as the new ramet leaf buds (RLB), and the new active axillary stolon buds (ASB) differentiated continually after the differentiation of the first leaf. Using the tandem mass tags (TMT) labeling method, a total of 7271 strawberry proteins were identified. Between ASB and DSB, the spliceosome DEPs, such as Ser/Arg-rich (SR) and heterogeneous nuclear ribonucleoprotein particle (hnRNP), showed the highest enrichment and high PPI connectivity. This indicated that the differences in DEPs (e.g., SF-3A and PK) at the transcriptional level may be causing the differences between the physiological statuses of ASB and DSB. As expected, the photosynthetic pre-form RLB mainly differentiated from ASB and DSB judging by the DEP enrichment of photosynthesis. However, there are still other specialized features of DEPs between RLB and DSB and between ASB and DSB. The DEPs relative to DNA duplication [e.g., minichromosome maintenance protein (MCM 2, 3, 4, 7)], provide a strong hint of functional gene duplication leading the bud heterogeneity between RLB and DSB. In addition, the top fold change DEP of LSH 10-like might be involved in the degeneration of SAM into RLBs, based on its significant function in modulating the plant shoot initiation. As for RLB/ASB, the phenylpropanoid biosynthesis pathway probably regulates the ramet axillary bud specialization, and further promotes the differentiation of xylem when ASB develops into a new stolon [e.g., cinnamyl alcohol dehydrogenase 1 (CAD1) and phenylalanine ammonia-lyase 1 (PAL1)].ConclusionsBy using phenotypic observation combined with proteomic networks with different types of strawberry stolon buds, the definite dormancy phase of DSB was identified, and the biological pathways and gene networks that might be responsible for heterogeneity among different stolon buds in strawberry were also revealed.

Different as night and day: Behavioural and life history responses to varied photoperiods in Daphnia magna.

Nearly all animal species have utilized photoperiod to cue seasonal behaviours and life history traits. We investigated photoperiod responses in keystone species, Daphnia magna, to identify molecular processes underlying ecologically important behaviours and traits using functional transcriptomic analyses. Daphnia magna were photoperiod‐entrained immediately posthatch to a standard control photoperiod of 16 light/ 8 dark hours (16L:8D) relative to shorter (4L:20D, 8L:16D, 12L:12L) and longer (20L:4D) day length photoperiods. Short‐day photoperiods induced significantly increased light‐avoidance behaviours relative to controls. Correspondingly, significant differential transcript expression for genes involved in glutamate signalling was observed, a critical signalling pathway in arthropod light‐avoidance behaviour. Additionally, period circadian protein and proteins coding F‐box/LRR‐repeat domains were differentially expressed which are recognized to establish circadian rhythms in arthropods. Indicators of metabolic rate increased in short‐day photoperiods which corresponded with broadscale changes in transcriptional expression across system‐level energy metabolism pathways. The most striking observations included significantly decreased neonate production at the shortest day length photoperiod (4L:20D) and significantly increased male production across short‐day and equinox photoperiods (4L:20D, 8L:16D and 12L:12D). Transcriptional expression consistent with putative mechanisms of male production was observed including photoperiod‐dependent expression of transformer‐2 sex‐determining protein and small nuclear ribonucleoprotein particles (snRNPs) which control splice variant expression for genes like transformer. Finally, increased transcriptional expression of glutamate has also been shown to induce male production in Daphnia pulex via photoperiod‐sensitive mechanisms. Overall, photoperiod entrainment affected molecular pathways that underpin critical behavioural and life history traits in D. magna providing fundamental insights into biological responses to this primary environmental cue.

Precursor RNA processing 3 is required for male fertility, and germline stem cell self-renewal and differentiation via regulating spliceosome function in Drosophila testes

The nuclear pre-mRNA spliceosome is a large complex containing five small nuclear ribonucleoprotein particles (snRNPs) and many splicing factors. Messenger RNAs (mRNAs) are generated from pre-mRNAs by the process of RNA splicing, which is conserved in eukaryotes. Precursor RNA processing 3 (Prp3) is a U4/U6-associated snRNP whose function remains largely unknown. In the present study, using genetic manipulation of a Drosophila melanogaster testis model, we demonstrated that Prp3 is essential for male fertility in Drosophila. Prp3 deficiency in germline stem cells (GSCs) and early cyst cells resulted in abnormal structure of testes and maintenance defects of GSCs and cyst stem cells. Knockdown of Prp3 in spermatogonia and early cyst cells mediated tumor formation caused by differentiation defects. Using an in vitro assay, knockdown of Prp3 decreased proliferation and increased cell death, and controlled the spliceosome function via regulating spliceosome subunits expression in Drosophila S2 cells. We also identified two other splicing factors in the Prp complex (Prp19 and Prp8), which mimicked the phenotype of Prp3 in the Drosophila stem cell niche. Our results revealed a significant role of precursor RNA processing factors in male testes, indicating that Prp3, a key spliceosome component in the Prp complex, is essential for male fertility, and germline stem cell self-renewal and differentiation, via regulating the spliceosome function in Drosophila testes.

Mouse models of human craniofacials spliceosomopathies: Are they neurocristopathies?

Splicing, the removal of introns from pre‐messenger RNA is an essential step for expression of most genes in multicellular organisms and for expanding the number of proteins coded by genomes. Recently, exome sequencing revealed that mutations in splicing factors and small nuclear ribonucleoprotein particles (snRNPs) which form core components of the spliceosome, are responsible for craniofacial malformations. Our group used in situ hybridization to examine expression of three core components of the spliceosome: Eftud2 – mutated in patients with mandibulofacial dysostosis, Guion‐Almeida type (MFDGA); SnrpB – responsible for Cerebrocostomandibular Syndrome (CMS); and Sf3b4 which is responsible for Nager syndrome. Herein, we report expression of Eftud2, Sf3b4 and SnrpB during craniofacial development. In addition, we used CRISPR/Cas9 to generate mice with deletion (Eftud2 del) and conditional mutation of exon 2 of Eftud2 (Eftud2 flox). At embryonic days (E) 7.5 and 8.5, Eftud2 was highly expressed in ectodermal and mesodermal components of the future craniofacial region, by E9.5 expression was also found in the body wall and developing heart. Eftud2 del heterozygous mice were viable and fertile, though these embryos showed reduced levels of Eftud2 mRNA and protein. In contrast, Eftud2 del homozygous mutant embryos arrest at E3.5 and failed to grow and hatch ex vivo. To examine the requirement for Eftud2 in neural crest cells, the Wnt1‐Cre2 transgenic line was used to delete exon 2 of Eftud2 specifically in that lineage. Eftud2 flox homozygous mutant embryos carrying the Wnt1‐Cre2 transgene displayed hypoplasia of the midbrain and pharyngeal arches starting at E9.5. By E11.5, most embryos also had an open neural tube and all embryos showed exencephaly at E14.5. Cartilage preparations revealed an absence of cartilage in the head, reduction/or absence of Meckel's cartilage, and abnormal inner ear development. Since deletion of exon 2 is predicted to generate a truncated protein with partial function, our data suggest that normal levels of Eftud2 is crucial in neural crest cells for normal craniofacial development. Future studies are focused on elucidating the molecular and transcriptional basis of MFDM using this mouse model.Support or Funding InformationCanadian Institute of Health Research (MOP#142452)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interpreting the Dynamics of Binding Interactions of snRNA and U1A Using a Coarse-Grained Model

The binding interactions of small nuclear RNAs (snRNA) and the associated protein factors are critical to the function of spliceosomes in alternatively splicing primary RNA transcripts. Although molecular dynamics simulations are a powerful tool to interpret the mechanism of biological processes, the atomic-level simulations are, however, too expensive and with limited accuracy for the large-size systems, such as snRNA-protein complexes. We extend the coarse-grained Gaussian network model, which models the RNA-protein complexes as a harmonic chain of Cα, P, and O4′ atoms, to investigating the impact of the snRNA-binding interaction on the dynamic stability of the human U1A protein, which is a major component of the spliceosomal U1 small nuclear ribonucleoprotein particle. The results reveal that the first and third loops and the C-terminal helix regions of the U1A domain undergo a significant loss of flexibility upon the RNA binding due to the forming of mostly electrostatic and hydrogen bond interactions with RNA 5′ stem and loop. By examining the residues whose mutations significantly change the binding free energy between U1A and snRNA, the Gaussian network model-based calculations show that not only the residues at the binding sites that are traditionally considered to play a major role in U1A-RNA association but also those residues that are far away from the RNA-binding interface can participate in the long-range allosteric signal transmission; these calculations are quantitatively consistent with the data observed in the recent snRNA binding experiments. The study demonstrates a useful avenue to utilize the simplified elastic network model to investigate the dynamics characteristics of the biologically important macromolecular interactions.

Mixed connective tissue disease: state of the art on clinical practice guidelines

Mixed connective tissue disease (MCTD) is a complex overlap disease with features of different autoimmune connective tissue diseases (CTDs) namely systemic sclerosis, poly/dermatomyositis and systemic lupus erythematous in patients with...

Prespliceosome structure provides insights into spliceosome assembly and regulation

The spliceosome catalyzes the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors1. For branching, the intron 5'-splice site (5'SS) and the branch point (BP) sequence are selected and brought into the prespliceosome by the U1 and U2 snRNPs1, which is a focal point for the regulation by alternative splicing factors2. The U4/U6.U5 tri-snRNP subsequently joins the prespliceosome to form the complete pre-catalytic spliceosome. Recent studies have revealed the structural basis of the branching and exon-ligation reactions3. However, the structural basis of early spliceosome assembly events remains poorly understood4. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae prespliceosome at near-atomic resolution. The structure reveals an induced stabilization of the 5'SS in the U1 snRNP, and provides structural insights into the functions of the human alternative splicing factors LUC7-like (yeast Luc7) and TIA-1 (yeast Nam8) that are linked to human disease5,6. In the prespliceosome, the U1 snRNP associates with the U2 snRNP through a stable contact with the U2 3' domain and a transient yeast-specific contact with the U2 SF3b-containing 5' region, leaving its tri-snRNP-binding interface fully exposed. The results suggest mechanisms for 5'SS transfer to the U6 ACAGAGA region within the assembled spliceosome and for its subsequent conversion to the activation-competent B complex spliceosome7,8. Taken together, the data provide a working model to investigate the early steps of spliceosome assembly.

Cryo-EM Studies of Pre-mRNA Splicing: From Sample Preparation to Model Visualization.

The removal of noncoding introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.

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

Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes

SummaryPat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5′ to 3′ mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3′ UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation.

Specialized assembly factors facilitate the formation of many macromolecular complexes in vivo. The formation of Sm core structures of spliceosomal U-rich small nuclear ribonucleoprotein particles (UsnRNPs) requires assembly factors united in protein arginine methyltransferase 5 (PRMT5) and survival motor neuron (SMN) complexes. We demonstrate that perturbations of this assembly machinery trigger complex cellular responses that prevent aggregation of unassembled Sm proteins. Inactivation of the SMN complex results in the initial tailback of Sm proteins on the PRMT5 complex, followed by down-regulation of their encoding mRNAs. In contrast, reduction of pICln, a PRMT5 complex subunit, leads to the retention of newly synthesized Sm proteins on ribosomes and their subsequent lysosomal degradation. Overexpression of Sm proteins under these conditions results in a surplus of Sm proteins over pICln, promoting their aggregation. Our studies identify an elaborate safeguarding system that prevents individual Sm proteins from aggregating, contributing to cellular UsnRNP homeostasis.

Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) asa major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.

Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones.

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.