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
• oční proteiny genetika   metabolismus   MeSH
• prekurzory RNA * metabolismus   genetika   MeSH
• proteiny vázající RNA metabolismus   genetika   MeSH
• retinální pigmentový epitel metabolismus   patologie   MeSH
• retinopathia pigmentosa * genetika   metabolismus   patologie   MeSH
• ribonukleoproteiny malé jaderné metabolismus   genetika   MeSH
• RNA malá jaderná genetika   metabolismus   MeSH
• sestřih RNA * genetika   MeSH
• spliceozomy metabolismus   genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U4-U6 MeSH
• oční proteiny MeSH
• prekurzory RNA * MeSH
• proteiny vázající RNA MeSH
• PRPF8 protein, human MeSH Prohlížeč
• ribonukleoproteiny malé jaderné MeSH
• RNA malá jaderná MeSH
• SNRNP200 protein, human MeSH Prohlížeč

This study aims to characterize the proteome composition of organ-derived protein extracts from rabbits. Protein isolation was performed using soft homogenization and size exclusion via ultrafiltration. The proteome analysis of the ultrafiltrates was conducted using gel electrophoresis, and the mass spectrometry data were subjected to gene ontology analysis. Proteomic profiling revealed comprehensive protein profiles associated with RNA regulation, fatty acid binding, inflammatory response, oxidative stress, and metabolism. Additionally, our results demonstrate the presence of abundant small proteins, as observed in the mass spectrometry datasets. Small proteins and peptides are crucial in transcription modulation and various biological processes. The protein networks identified in the ultrafiltrates have the potential to enhance and complement biological therapeutic interventions. Data are available via ProteomeXchange with identifier PXD050039.

• Klíčová slova
• organ specificity, proteomics, small proteins, ultrafiltration,
• MeSH
• hmotnostní spektrometrie MeSH
• králíci MeSH
• peptidy MeSH
• proteom * metabolismus   MeSH
• proteomika * metody   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy MeSH
• proteom * MeSH

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

• Klíčová slova
• Development, Genetic diseases, Genetics, Neurodevelopment, iPS cells,
• MeSH
• enzymy opravy DNA genetika   MeSH
• genové regulační sítě MeSH
• jaderné proteiny genetika   MeSH
• lidé MeSH
• missense mutace MeSH
• neurovývojové poruchy * genetika   MeSH
• sestřih RNA MeSH
• sestřihové faktory genetika   MeSH
• spliceozomy * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• enzymy opravy DNA MeSH
• jaderné proteiny MeSH
• PRPF19 protein, human MeSH Prohlížeč
• sestřihové faktory MeSH

RNA capping is a prominent RNA modification that influences RNA stability, metabolism, and function. While it was long limited to the study of the most abundant eukaryotic canonical m7G cap, the field recently went through a large paradigm shift with the discovery of non-canonical RNA capping in bacteria and ultimately all domains of life. The repertoire of non-canonical caps has expanded to encompass metabolite caps, including NAD, FAD, CoA, UDP-Glucose, and ADP-ribose, alongside alarmone dinucleoside polyphosphate caps, and methylated phosphate cap-like structures. This review offers an introduction into the field, presenting a summary of the current knowledge about non-canonical RNA caps. We highlight the often still enigmatic biological roles of the caps together with their processing enzymes, focusing on the most recent discoveries. Furthermore, we present the methods used for the detection and analysis of these non-canonical RNA caps and thus provide an introduction into this dynamic new field.

• Klíčová slova
• NAD, RNA, RNA cap, RNA modifications, dinucleoside polyphosphate, epitranscriptomics,
• MeSH
• Bacteria genetika   metabolismus   MeSH
• lidé MeSH
• RNA čepičky * metabolismus   chemie   MeSH
• RNA chemie   metabolismus   genetika   MeSH
• stabilita RNA MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• RNA čepičky * MeSH
• RNA MeSH

Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies, including multiple myeloma (MM). However, this plasma cell malignancy remains incurable, and novel therapies are therefore urgently needed. Whole-genome transcriptome analyses in a large cohort of MM patients demonstrated that alterations in pre-mRNA splicing (AS) are frequent in MM. This manuscript describes approaches to identify disease-specific alterations in MM and proposes RNA-based therapeutic strategies to eradicate such alterations. As a "proof of concept", we examined the causes of aberrant HMMR (Hyaluronan-mediated motility receptor) splicing in MM. We identified clusters of single nucleotide variations (SNVs) in the HMMR transcript where the altered splicing took place. Using bioinformatics tools, we predicted SNVs and splicing factors that potentially contribute to aberrant HMMR splicing. Based on bioinformatic analyses and validation studies, we provided the rationale for RNA-based therapeutic strategies to selectively inhibit altered HMMR splicing in MM. Since splicing is a hallmark of many cancers, strategies described herein for target identification and the design of RNA-based therapeutics that inhibit gene splicing can be applied not only to other genes in MM but also more broadly to other hematological malignancies and solid tumors as well.

• MeSH
• alternativní sestřih MeSH
• hematologické nádory * MeSH
• lidé MeSH
• mnohočetný myelom * genetika   terapie   MeSH
• RNA MeSH
• sestřih RNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• RNA MeSH

Many nascent long non-coding RNAs (lncRNAs) undergo the same maturation steps as pre-mRNAs of protein-coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences using the ncRNA-a2 as a model. Genome-wide analyses of intergenic lncRNAs (lincRNAs) revealed that lincRNA splicing efficiency positively correlates with 5'ss strength while no such correlation was identified for PCGs. In addition, efficiently spliced lincRNAs have higher thymidine content in the polypyrimidine tract (PPT) compared to efficiently spliced PCGs. Using model lincRNAs, we provide experimental evidence that strengthening the 5'ss and increasing the T content in PPT significantly enhances lincRNA splicing. We further showed that lincRNA exons contain less putative binding sites for SR proteins. To map binding of SR proteins to lincRNAs, we performed iCLIP with SRSF2, SRSF5 and SRSF6 and analyzed eCLIP data for SRSF1, SRSF7 and SRSF9. All examined SR proteins bind lincRNA exons to a much lower extent than expression-matched PCGs. We propose that lincRNAs lack the cooperative interaction network that enhances splicing, which renders their splicing outcome more dependent on the optimality of splice sites.

• MeSH
• HeLa buňky MeSH
• introny * MeSH
• lidé MeSH
• místa sestřihu RNA * MeSH
• pyrimidiny analýza   MeSH
• RNA dlouhá nekódující metabolismus   MeSH
• serin-arginin sestřihové faktory metabolismus   MeSH
• sestřih RNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• místa sestřihu RNA * MeSH
• pyrimidine MeSH Prohlížeč
• pyrimidiny MeSH
• RNA dlouhá nekódující MeSH
• serin-arginin sestřihové faktory 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

Trans-splicing is a process by which 5'- and 3'-ends of two pre-RNA molecules transcribed from different sites of the genome can be joined together to form a single RNA molecule. The spliced leader (SL) trans-splicing is mediated by the spliceosome and it allows the replacement of 5'-end of pre-mRNA by 5'(SL)-end of SL-RNA. This form of splicing has been observed in many phylogenetically unrelated eukaryotes. Either the SL trans-splicing (SLTS) originated in the last eukaryotic common ancestor (LECA) (or even earlier) and it was lost in most eukaryotic lineages, or this mechanism of RNA processing evolved several times independently in various unrelated eukaryotic taxa. The bioinformatic comparisons of SL-RNAs from various eukaryotic taxonomic groups have revealed the similarities of secondary structures of most SL-RNAs and a relative conservation of their splice sites (SSs) and Sm-binding sites (SmBSs). We propose that such structural and functional similarities of SL-RNAs are unlikely to have evolved repeatedly many times. Hence, we favor the scenario of an early evolutionary origin for the SLTS and multiple losses of SL-RNAs in various eukaryotic lineages.

• Klíčová slova
• Intron, RNA secondary structure, SL-RNA, Sm-binding site, Spliceosome,
• MeSH
• Eukaryota genetika   metabolismus   MeSH
• fylogeneze MeSH
• molekulární evoluce * MeSH
• prekurzory RNA metabolismus   MeSH
• RNA se sestřihovou vedoucí sekvencí genetika   metabolismus   MeSH
• trans-splicing * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• prekurzory RNA MeSH
• RNA se sestřihovou vedoucí sekvencí 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

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
• Cajalova tělíska metabolismus   MeSH
• genetická transkripce MeSH
• lidé MeSH
• posttranskripční úpravy RNA MeSH
• ribonukleoproteiny malé jaderné genetika   metabolismus   MeSH
• spliceozomy MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ribonukleoproteiny malé jaderné MeSH