Retinitis pigmentosa (RP) is a hereditary disorder caused by mutations in more than 70 different genes including those that encode proteins important for pre-mRNA splicing. Most RP-associated mutations in splicing factors reduce either their expression, stability or incorporation into functional splicing complexes. However, we have previously shown that two RP mutations in PRPF8 (F2314L and Y2334N) and two in SNRNP200 (S1087L and R1090L) behaved differently, and it was still unclear how these mutations affect the functions of both proteins. To investigate this in the context of functional spliceosomes, we used iCLIP in HeLa and retinal pigment epithelial (RPE) cells. We found that both mutations in the RNA helicase SNRNP200 change its interaction with U4 and U6 snRNAs. The significantly broader binding profile of mutated SNRNP200 within the U4 region upstream of the U4/U6 stem I strongly suggests that its activity to unwind snRNAs is impaired. This was confirmed by FRAP measurements and helicase activity assays comparing mutant and WT protein. The RP variants of PRPF8 did not affect snRNAs, but showed a reduced binding to pre-mRNAs, which resulted in the slower splicing of introns and altered expression of hundreds of genes in RPE cells. This suggests that changes in the expression and splicing of specific genes are the main driver of retinal degeneration in PRPF8-linked RP.

• Keywords
• PRPF8, Pre-mRNA splicing, Retinitis pigmentosa, SNRNP200, iCLIP,
• MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   genetics   MeSH
• Mutation * MeSH
• RNA Precursors * metabolism   genetics   MeSH
• RNA-Binding Proteins * metabolism   genetics   MeSH
• Retinal Pigment Epithelium metabolism   MeSH
• Retinitis Pigmentosa * genetics   metabolism   pathology   MeSH
• Ribonucleoproteins, Small Nuclear * metabolism   genetics   MeSH
• RNA, Small Nuclear * metabolism   genetics   MeSH
• RNA Splicing genetics   MeSH
• RNA Splicing Factors metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ribonucleoprotein, U4-U6 Small Nuclear MeSH
• RNA Precursors * MeSH
• RNA-Binding Proteins * MeSH
• PRPF8 protein, human MeSH Browser
• Ribonucleoproteins, Small Nuclear * MeSH
• RNA, Small Nuclear * MeSH
• RNA Splicing Factors MeSH
• SNRNP200 protein, human MeSH Browser

The nucleus of higher eukaryotes contains a number of structures that concentrate specific biomolecules and play distinct roles in nuclear metabolism. In recent years, the molecular mechanisms controlling their formation have been intensively studied. In this brief review, I focus on coilin and Cajal bodies. Coilin is a key scaffolding protein of Cajal bodies that is evolutionarily conserved in metazoans. Cajal bodies are thought to be one of the archetypal nuclear structures involved in the metabolism of several short non-coding nuclear RNAs. Yet surprisingly little is known about the structure and function of coilin, and a comprehensive model to explain the origin of Cajal bodies is also lacking. Here, I summarize recent results on Cajal bodies and coilin and discuss them in the context of the last three decades of research in this field.

• Keywords
• Cajal bodies, coilin, snRNA, snRNP, snoRNA, telomerase RNA,
• MeSH
• Cell Nucleus * MeSH
• Coiled Bodies * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

A subset of patients with retinitis pigmentosa (RP) carry mutations in several spliceosomal components including the PRPF8 protein. Here, we established two alleles of murine Prpf8 that genocopy or mimic aberrant PRPF8 found in RP patients-the substitution p.Tyr2334Asn and an extended protein variant p.Glu2331ValfsX15. Homozygous mice expressing the aberrant Prpf8 variants developed within the first 2 mo progressive atrophy of the cerebellum because of extensive granule cell loss, whereas other cerebellar cells remained unaffected. We further show that a subset of circRNAs were deregulated in the cerebellum of both Prpf8-RP mouse strains. To identify potential risk factors that sensitize the cerebellum for Prpf8 mutations, we monitored the expression of several splicing proteins during the first 8 wk. We observed down-regulation of all selected splicing proteins in the WT cerebellum, which coincided with neurodegeneration onset. The decrease in splicing protein expression was further pronounced in mouse strains expressing mutated Prpf8. Collectively, we propose a model where physiological reduction in spliceosomal components during postnatal tissue maturation sensitizes cells to the expression of aberrant Prpf8 and the subsequent deregulation of circRNAs triggers neuronal death.

• MeSH
• RNA, Circular MeSH
• Cerebellum MeSH
• Mutation MeSH
• Mice MeSH
• RNA-Binding Proteins * genetics   MeSH
• Retinitis Pigmentosa * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Circular MeSH
• RNA-Binding Proteins * MeSH

Retinitis pigmentosa (RP) is a hereditary disease affecting tens of thousands of people world-wide. Here we analyzed the effect of an amino acid substitution in the RNA helicase DHX38 (Prp16) causing RP. DHX38 has been proposed as the helicase important for the 2nd step of splicing. We showed that DHX38 associates with key splicing factors involved in both splicing steps but did not find any evidence that the RP mutations changes DHX38 interaction profile with the spliceosome. We further downregulated DHX38 and monitored changes in splicing. We observed only minor perturbations of general splicing but detected modulation of ~70 alternative splicing events. Next, we probed DHX38 function in splicing of retina specific genes and found that FSCN2 splicing is dependent on DHX38. In addition, RHO splicing was inhibited specifically by expression of DHX38 RP variant. Finally, we showed that overexpression of DHX38 promotes usage of canonical as well as cryptic 5' splice sites in HBB splicing reporter. Together, our data show that DHX38 is a splicing factor that promotes splicing of cryptic splice sites and regulate alternative splicing. We further provide evidence that the RP-linked substitution G332D modulates DHX38 splicing activity.

• MeSH
• DEAD-box RNA Helicases * genetics   MeSH
• Humans MeSH
• RNA Splice Sites MeSH
• Mutation MeSH
• Retinitis Pigmentosa * genetics   MeSH
• RNA Splicing MeSH
• RNA Splicing Factors * genetics   MeSH
• Spliceosomes metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DEAD-box RNA Helicases * MeSH
• DHX38 protein, human MeSH Browser
• RNA Splice Sites MeSH
• RNA Splicing Factors * MeSH

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
• Names of Substances
• EFTUD2 protein, human MeSH Browser
• Peptide Elongation Factors MeSH
• DNA Repair Enzymes MeSH
• Nuclear Proteins MeSH
• Ribonucleoprotein, U5 Small Nuclear MeSH
• Tumor Suppressor Proteins MeSH
• RNA-Binding Proteins MeSH
• PRPF19 protein, human MeSH Browser
• PRPF6 protein, human MeSH Browser
• PRPF8 protein, human MeSH Browser
• Recombinant Fusion Proteins MeSH
• Ribonucleoproteins, Small Nuclear MeSH
• RNA Splicing Factors MeSH
• SNRNP200 protein, human MeSH Browser
• Transcription Factors MeSH
• TSSC4 protein, human MeSH Browser

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.

• MeSH
• Exoribonucleases metabolism   MeSH
• HeLa Cells MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Organelles metabolism   MeSH
• SMN Complex Proteins metabolism   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• RNA, Small Nuclear chemistry   metabolism   MeSH
• Base Sequence MeSH
• RNA Stability MeSH
• RNA Transport * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DIS3L2 protein, human MeSH Browser
• Exoribonucleases MeSH
• GEMIN5 protein, human MeSH Browser
• LSM1 protein, human MeSH Browser
• SMN Complex Proteins MeSH
• RNA-Binding Proteins MeSH
• Proto-Oncogene Proteins MeSH
• RNA, Small Nuclear 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
• Names of Substances
• Ribonucleoprotein, U2 Small Nuclear MeSH
• RNA, Small Nuclear MeSH
• U2 small nuclear RNA MeSH Browser