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

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

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
• elongační faktory genetika   metabolismus   MeSH
• HeLa buňky MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U1 metabolismus   MeSH
• malý jaderný ribonukleoprotein U4-U6 metabolismus   MeSH
• malý jaderný ribonukleoprotein U5 genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• multiproteinové komplexy MeSH
• mutace MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• proteiny vázající vápník metabolismus   MeSH
• proteomika metody   MeSH
• retinopathia pigmentosa genetika   metabolismus   MeSH
• RNA interference MeSH
• sestřih RNA * MeSH
• stabilita proteinů MeSH
• transfekce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• EFTUD2 protein, human MeSH Prohlížeč
• elongační faktory MeSH
• malý jaderný ribonukleoprotein U1 MeSH
• malý jaderný ribonukleoprotein U4-U6 MeSH
• malý jaderný ribonukleoprotein U5 MeSH
• messenger RNA MeSH
• multiproteinové komplexy MeSH
• prekurzory RNA MeSH
• proteiny tepelného šoku HSP90 MeSH
• proteiny vázající RNA MeSH
• proteiny vázající vápník MeSH
• PRPF8 protein, human MeSH Prohlížeč
• TESC protein, human MeSH Prohlížeč

A majority of human genes contain non-coding intervening sequences - introns that must be precisely excised from the pre-mRNA molecule. This event requires the coordinated action of five major small nuclear ribonucleoprotein particles (snRNPs) along with additional non-snRNP splicing proteins. Introns must be removed with nucleotidal precision, since even a single nucleotide mistake would result in a reading frame shift and production of a non-functional protein. Numerous human inherited diseases are caused by mutations that affect splicing, including mutations in proteins which are directly involved in splicing catalysis. One of the most common hereditary diseases associated with mutations in core splicing proteins is retinitis pigmentosa (RP). So far, mutations in more than 70 genes have been connected to RP. While the majority of mutated genes are expressed specifically in the retina, eight target genes encode for ubiquitous core snRNP proteins (Prpf3, Prpf4, Prpf6, Prpf8, Prpf31, and SNRNP200/Brr2) and splicing factors (RP9 and DHX38). Why mutations in spliceosomal proteins, which are essential in nearly every cell in the body, causes a disease that displays such a tissue-specific phenotype is currently a mystery. In this review, we recapitulate snRNP functions, summarize the missense mutations which are found in spliceosomal proteins as well as their impact on protein functions and discuss specific models which may explain why the retina is sensitive to these mutations.

• Klíčová slova
• Retinitis pigmentosa, snRNP, splicing,
• MeSH
• introny MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• missense mutace MeSH
• myši MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• retinopathia pigmentosa genetika   MeSH
• ribonukleoproteiny malé jaderné genetika   metabolismus   MeSH
• sestřih RNA MeSH
• sestřihové faktory genetika   metabolismus   MeSH
• spliceozomy genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• prekurzory RNA MeSH
• ribonukleoproteiny malé jaderné MeSH
• sestřihové faktory MeSH