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.

• Klíčová slova
• PRPF8, Pre-mRNA splicing, Retinitis pigmentosa, SNRNP200, iCLIP,
• MeSH
• HeLa buňky MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U4-U6 metabolismus   genetika   MeSH
• mutace * MeSH
• prekurzory RNA * metabolismus   genetika   MeSH
• proteiny vázající RNA * metabolismus   genetika   MeSH
• retinální pigmentový epitel metabolismus   MeSH
• retinopathia pigmentosa * genetika   metabolismus   patologie   MeSH
• ribonukleoproteiny malé jaderné * metabolismus   genetika   MeSH
• RNA malá jaderná * metabolismus   genetika   MeSH
• sestřih RNA genetika   MeSH
• sestřihové faktory metabolismus   genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U4-U6 MeSH
• prekurzory RNA * MeSH
• proteiny vázající RNA * MeSH
• PRPF8 protein, human MeSH Prohlížeč
• ribonukleoproteiny malé jaderné * MeSH
• RNA malá jaderná * MeSH
• sestřihové faktory MeSH
• SNRNP200 protein, human MeSH Prohlížeč

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.

• Klíčová slova
• Cajal bodies, coilin, snRNA, snRNP, snoRNA, telomerase RNA,
• MeSH
• buněčné jádro * MeSH
• Cajalova tělíska * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy 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
• kruhová RNA MeSH
• mozeček MeSH
• mutace MeSH
• myši MeSH
• proteiny vázající RNA * genetika   MeSH
• retinopathia pigmentosa * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kruhová RNA MeSH
• proteiny vázající RNA * 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-helikasy * genetika   MeSH
• lidé MeSH
• místa sestřihu RNA MeSH
• mutace MeSH
• retinopathia pigmentosa * genetika   MeSH
• sestřih RNA MeSH
• sestřihové faktory * genetika   MeSH
• spliceozomy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DEAD-box RNA-helikasy * MeSH
• DHX38 protein, human MeSH Prohlížeč
• místa sestřihu RNA MeSH
• sestřihové faktory * 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 regulace MeSH
• elongační faktory MeSH
• enzymy opravy DNA metabolismus   MeSH
• HeLa buňky MeSH
• interakční proteinové domény a motivy MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U5 chemie   metabolismus   MeSH
• nádorové supresorové proteiny chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• proteiny vázající RNA metabolismus   MeSH
• rekombinantní fúzní proteiny MeSH
• ribonukleoproteiny malé jaderné chemie   metabolismus   MeSH
• sestřih RNA MeSH
• sestřihové faktory metabolismus   MeSH
• spliceozomy metabolismus   MeSH
• transkripční faktory MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EFTUD2 protein, human MeSH Prohlížeč
• elongační faktory MeSH
• enzymy opravy DNA MeSH
• jaderné proteiny MeSH
• malý jaderný ribonukleoprotein U5 MeSH
• nádorové supresorové proteiny MeSH
• proteiny vázající RNA MeSH
• PRPF19 protein, human MeSH Prohlížeč
• PRPF6 protein, human MeSH Prohlížeč
• PRPF8 protein, human MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH
• ribonukleoproteiny malé jaderné MeSH
• sestřihové faktory MeSH
• SNRNP200 protein, human MeSH Prohlížeč
• transkripční faktory MeSH
• TSSC4 protein, human MeSH Prohlížeč

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
• exoribonukleasy metabolismus   MeSH
• HeLa buňky MeSH
• konformace nukleové kyseliny * MeSH
• lidé MeSH
• organely metabolismus   MeSH
• proteinový komplex SMN metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• RNA malá jaderná chemie   metabolismus   MeSH
• sekvence nukleotidů MeSH
• stabilita RNA MeSH
• transport RNA * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DIS3L2 protein, human MeSH Prohlížeč
• exoribonukleasy MeSH
• GEMIN5 protein, human MeSH Prohlížeč
• LSM1 protein, human MeSH Prohlížeč
• proteinový komplex SMN MeSH
• proteiny vázající RNA MeSH
• protoonkogenní proteiny MeSH
• RNA malá jaderná 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
• buněčné jádro genetika   MeSH
• Cajalova tělíska genetika   metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U2 genetika   MeSH
• regulace genové exprese genetika   MeSH
• RNA malá jaderná genetika   MeSH
• spliceozomy genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U2 MeSH
• RNA malá jaderná MeSH
• U2 small nuclear RNA MeSH Prohlížeč