U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo 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 the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a 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.

• MeSH
• Down-Regulation MeSH
• Peptide Elongation Factors MeSH
• DNA Repair Enzymes metabolism   MeSH
• HeLa Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• Nuclear Proteins metabolism   MeSH
• Humans MeSH
• Ribonucleoprotein, U5 Small Nuclear chemistry   metabolism   MeSH
• Tumor Suppressor Proteins chemistry   genetics   metabolism   MeSH
• Protein Domains MeSH
• RNA-Binding Proteins metabolism   MeSH
• Recombinant Fusion Proteins MeSH
• Ribonucleoproteins, Small Nuclear chemistry   metabolism   MeSH
• RNA Splicing MeSH
• RNA Splicing Factors metabolism   MeSH
• Spliceosomes metabolism   MeSH
• Transcription Factors MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The U4/U6·U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) is an essential pre-mRNA splicing factor, which is assembled in a stepwise manner before each round of splicing. It was previously shown that the tri-snRNP is formed in Cajal bodies (CBs), but little is known about the dynamics of this process. Here we created a mathematical model of tri-snRNP assembly in CBs and used it to fit kinetics of individual snRNPs monitored by fluorescence recovery after photobleaching. A global fitting of all kinetic data determined key reaction constants of tri-snRNP assembly. Our model predicts that the rates of di-snRNP and tri-snRNP assemblies are similar and that ∼230 tri-snRNPs are assembled in one CB per minute. Our analysis further indicates that tri-snRNP assembly is approximately 10-fold faster in CBs than in the surrounding nucleoplasm, which is fully consistent with the importance of CBs for snRNP formation in rapidly developing biological systems. Finally, the model predicted binding between SART3 and a CB component. We tested this prediction by Förster resonance energy transfer and revealed an interaction between SART3 and coilin in CBs.

• MeSH
• Antigens, Neoplasm genetics   metabolism   MeSH
• Cell Nucleus genetics   metabolism   MeSH
• Coiled Bodies genetics   metabolism   MeSH
• HeLa Cells MeSH
• Nuclear Proteins metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear genetics   metabolism   MeSH
• Ribonucleoprotein, U5 Small Nuclear genetics   metabolism   MeSH
• Models, Molecular MeSH
• Cell Line, Tumor MeSH
• RNA Precursors genetics   metabolism   MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Ribonucleoproteins, Small Nuclear genetics   metabolism   MeSH
• RNA Helicases genetics   metabolism   MeSH
• RNA Splicing genetics   MeSH
• Spliceosomes genetics   metabolism   MeSH
• Protein Binding genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Peptide Elongation Factors genetics   metabolism   MeSH
• HeLa Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Ribonucleoprotein, U1 Small Nuclear metabolism   MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   MeSH
• Ribonucleoprotein, U5 Small Nuclear genetics   metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Multiprotein Complexes MeSH
• Mutation MeSH
• RNA Precursors genetics   metabolism   MeSH
• HSP90 Heat-Shock Proteins metabolism   MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Calcium-Binding Proteins metabolism   MeSH
• Proteomics methods   MeSH
• Retinitis Pigmentosa genetics   metabolism   MeSH
• RNA Interference MeSH
• RNA Splicing * MeSH
• Protein Stability MeSH
• Transfection MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Split gene architecture of most human genes requires removal of intervening sequences by mRNA splicing that occurs on large multiprotein complexes called spliceosomes. Mutations compromising several spliceosomal components have been recorded in degenerative syndromes and haematological neoplasia, thereby highlighting the importance of accurate splicing execution in homeostasis of assorted adult tissues. Moreover, insufficient splicing underlies defective development of craniofacial skeleton and upper extremities. This review summarizes recent advances in the understanding of splicing factor function deduced from cryo-EM structures. We combine these data with the characterization of splicing factors implicated in hereditary or somatic disorders, with a focus on potential functional consequences the mutations may elicit in spliceosome assembly and/or performance. Given aberrant splicing or perturbations in splicing efficiency substantially underpin disease pathogenesis, profound understanding of the mis-splicing principles may open new therapeutic vistas. In three major sections dedicated to retinal dystrophies, hereditary acrofacial syndromes, and haematological malignancies, we delineate the noticeable variety of conditions associated with dysfunctional splicing and accentuate recurrent patterns in splicing defects.

• MeSH
• Cryoelectron Microscopy MeSH
• Protein Conformation MeSH
• Humans MeSH
• Mutation MeSH
• Disease genetics   MeSH
• RNA Precursors genetics   MeSH
• Ribonucleoproteins, Small Nuclear chemistry   genetics   ultrastructure   MeSH
• RNA Splicing * MeSH
• Spliceosomes genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

• MeSH
• Gliotoxin * metabolism   MeSH
• HeLa Cells MeSH
• HIV Infections * drug therapy   MeSH
• HIV-1 * metabolism   MeSH
• Humans MeSH
• Positive Transcriptional Elongation Factor B genetics   metabolism   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Ribonucleoproteins, Small Nuclear chemistry   MeSH
• Ribonucleoproteins MeSH
• Transcription Factors metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Spliceosomal snRNPs are complex particles that proceed through a fascinating maturation pathway. Several steps of this pathway are closely linked to nuclear non-membrane structures called Cajal bodies. In this review, I summarize the last 20 y of research in this field. I primarily focus on snRNP biogenesis, specifically on the steps that involve Cajal bodies. I also evaluate the contribution of the Cajal body in snRNP quality control and discuss the role of snRNPs in Cajal body formation.

• MeSH
• Coiled Bodies metabolism   MeSH
• Transcription, Genetic MeSH
• Humans MeSH
• RNA Processing, Post-Transcriptional MeSH
• Ribonucleoproteins, Small Nuclear genetics   metabolism   MeSH
• Spliceosomes MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

The AD29 mutation in HPRP31 belongs to a series of mutations that were initially linked with the autosomal dominant disorder retinitis pigmentosa (RP) type 11. The HPRP31 gene encodes the hPrp31 protein that specifically associates with spliceosomal small nuclear ribonucleoprotein particles (snRNPs). Despite intensive research, it is still unclear how the AD29 (Ala216Pro) mutation causes RP. In this study, we report that the expression of this mutant protein affects cell proliferation and alters the structure of nuclear Cajal bodies that are connected with snRNP metabolism. Interestingly, these effects can be reversed by the over-expression of the hPrp6 protein, a binding partner of hPrp31. Although Ala216 is not contained within the U4 or U5 snRNP interacting domains, we present several lines of evidence that demonstrate that the association between the AD29 mutant and snRNPs in the cell nucleus is significantly reduced. Finally, we show that the stability of the AD29 mutant is severely affected resulting in its rapid degradation. Taken together, our results indicate that the Ala216Pro mutation destabilizes the hPrp31 protein structure in turn reducing its interaction with snRNP binding partners and leading to its rapid degradation. These findings significantly impact our understanding of the molecular mechanisms underlying RP and suggest that the insufficiency of the functional hPrp31 protein combined with the potential cytotoxicity associated with the expression the AD29 mutant are at least partially causative of the RP phenotype.

• MeSH
• Coiled Bodies genetics   metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Eye Proteins genetics   chemistry   metabolism   MeSH
• Retinitis Pigmentosa genetics   metabolism   MeSH
• Ribonucleoproteins, Small Nuclear genetics   metabolism   MeSH
• Spliceosomes genetics   metabolism   MeSH
• Protein Stability MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Cell Nucleus genetics   MeSH
• Coiled Bodies genetics   metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ribonucleoprotein, U2 Small Nuclear genetics   MeSH
• Gene Expression Regulation genetics   MeSH
• RNA, Small Nuclear genetics   MeSH
• Spliceosomes genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.

• MeSH
• Biomarkers metabolism   MeSH
• Coiled Bodies metabolism   MeSH
• Financing, Organized MeSH
• HeLa Cells MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   MeSH
• Ribonucleoprotein, U5 Small Nuclear metabolism   MeSH
• Survival of Motor Neuron 1 Protein metabolism   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   MeSH
• Spliceosomes metabolism   MeSH
• Carrier Proteins metabolism   MeSH
• Check Tag
• Humans MeSH

Splicing in S. cerevisiae has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the AMA1 intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in prp45(1-169) cells. We also measured pre-mRNA accumulation of the meiotic MER2 gene, which depends on the expression of Mer1 factor for splicing. prp45(1-169) cells accumulated approximately threefold higher levels of MER2 pre-mRNA than WT cells only when splicing was induced. To monitor cotranscriptional splicing, we determined the presence of early spliceosome assembly factors and snRNP complexes along the ECM33 and ACT1 genes. We found that prp45(1-169) hampered the cotranscriptional recruitment of U2 and, to a larger extent, U5 and NTC, while the U1 profile was unaffected. The recruitment of Prp45(1-169) was impaired similarly to U5 snRNP and NTC. Our results imply that Prp45 is required for timely formation of complex A, prior to stable physical association of U5/NTC with the emerging pre-mRNA substrate. We suggest that Prp45 facilitates conformational rearrangements and/or contacts that couple U1 snRNP-recognition to downstream assembly events.

• MeSH
• Introns MeSH
• Ribonucleoprotein, U1 Small Nuclear metabolism   MeSH
• Ribonucleoprotein, U2 Small Nuclear metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• RNA Splicing * MeSH
• Spliceosomes metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH