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

Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1-169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1-330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1-169), to the chromatin environment.

• Klíčová slova
• H2A.Z, Synthetic genetic array analysis, chromatin modifiers, co-transcriptional splicing, spliceosome assembly,
• MeSH
• fenotyp * MeSH
• histony metabolismus   genetika   MeSH
• introny * MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae - proteiny * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• sestřih RNA * MeSH
• sestřihové faktory genetika   metabolismus   MeSH
• spliceozomy * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• histony MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• sestřihové faktory MeSH

Pentacyclic triterpenoids, including ursolic acid (UA), are bioactive compounds with multiple biological activities involving anti-inflammatory effects. However, the mode of their action on mast cells, key players in the early stages of allergic inflammation, and underlying molecular mechanisms remain enigmatic. To better understand the effect of UA on mast cell signaling, here we examined the consequences of short-term treatment of mouse bone marrow-derived mast cells with UA. Using IgE-sensitized and antigen- or thapsigargin-activated cells, we found that 15 min exposure to UA inhibited high affinity IgE receptor (FcεRI)-mediated degranulation, calcium response, and extracellular calcium uptake. We also found that UA inhibited migration of mouse bone marrow-derived mast cells toward antigen but not toward prostaglandin E2 and stem cell factor. Compared to control antigen-activated cells, UA enhanced the production of tumor necrosis factor-α at the mRNA and protein levels. However, secretion of this cytokine was inhibited. Further analysis showed that UA enhanced tyrosine phosphorylation of the SYK kinase and several other proteins involved in the early stages of FcεRI signaling, even in the absence of antigen activation, but inhibited or reduced their further phosphorylation at later stages. In addition, we show that UA induced changes in the properties of detergent-resistant plasma membrane microdomains and reduced antibody-mediated clustering of the FcεRI and glycosylphosphatidylinositol-anchored protein Thy-1. Finally, UA inhibited mobility of the FcεRI and cholesterol. These combined data suggest that UA exerts its effects, at least in part, via lipid-centric plasma membrane perturbations, hence affecting the functions of the FcεRI signalosome.

• Klíčová slova
• immunoglobulin E, lipid raft, mast cell, plasma membrane, signal transduction, tumor necrosis factor, tyrosine kinase,
• MeSH
• antigeny metabolismus   MeSH
• degranulace buněk MeSH
• kyselina ursolová MeSH
• lipidy farmakologie   MeSH
• mastocyty metabolismus   MeSH
• myši MeSH
• receptory IgE * metabolismus   MeSH
• triterpeny * farmakologie   metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• lipidy MeSH
• receptory IgE * MeSH
• triterpeny * MeSH
• vápník 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

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.

• Klíčová slova
• BBSome, Bardet-Biedl Syndrome, Bardet-Biedl syndrome, assembly, ciliopathy, cilium, genetic disease, microscopic imaging, primary cilium, protein assembly, protein sorting,
• MeSH
• Bardetův-Biedlův syndrom genetika   metabolismus   patologie   MeSH
• buněčné linie MeSH
• cilie metabolismus   MeSH
• CRISPR-Cas systémy genetika   MeSH
• cytoplazma metabolismus   MeSH
• editace genu MeSH
• fluorescenční mikroskopie MeSH
• FRAP MeSH
• lidé MeSH
• mutace MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• proteiny asociované s mikrotubuly nedostatek   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Bbs1 protein, human MeSH Prohlížeč
• BBS4 protein, human MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny asociované s mikrotubuly MeSH

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3' splice sites (3'ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3'ss and branch points of a PUF60-dependent exon and the 3'ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3'ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

• MeSH
• aminokyselinové motivy MeSH
• exony * MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• heterogenní jaderné ribonukleoproteiny metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• krátké rozptýlené jaderné elementy MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U1 metabolismus   MeSH
• missense mutace * MeSH
• místa sestřihu RNA * MeSH
• proteiny vázající RNA metabolismus   MeSH
• represorové proteiny chemie   nedostatek   metabolismus   MeSH
• sekvenční analýza RNA MeSH
• sestřihové faktory chemie   nedostatek   metabolismus   MeSH
• sestřihový faktor U2AF MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HCC1 autoantigen MeSH Prohlížeč
• heterogenní jaderné ribonukleoproteiny MeSH
• jaderné proteiny MeSH
• malý jaderný ribonukleoprotein U1 MeSH
• místa sestřihu RNA * MeSH
• poly-U binding splicing factor 60KDa MeSH Prohlížeč
• proteiny vázající RNA MeSH
• represorové proteiny MeSH
• sestřihové faktory MeSH
• sestřihový faktor U2AF MeSH
• SNRNP70 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

There are a variety of complex metabolic processes ongoing simultaneously in the single, large mitochondrion of Trypanosoma brucei. Understanding the organellar environment and dynamics of mitochondrial proteins requires quantitative measurement in vivo. In this study, we have validated a method for immobilizing both procyclic stage (PS) and bloodstream stage (BS) T. brucei brucei with a high level of cell viability over several hours and verified its suitability for undertaking fluorescence recovery after photobleaching (FRAP), with mitochondrion-targeted yellow fluorescent protein (YFP). Next, we used this method for comparative analysis of the translational diffusion of mitochondrial RNA-binding protein 1 (MRP1) in the BS and in T. b. evansi. The latter flagellate is like petite mutant Saccharomyces cerevisiae because it lacks organelle-encoded nucleic acids. FRAP measurement of YFP-tagged MRP1 in both cell lines illuminated from a new perspective how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism. This work represents the first attempt to examine this process in live trypanosomes.

• MeSH
• mitochondriální proteiny genetika   MeSH
• mutace MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• RNA interference MeSH
• RNA mitochondriální MeSH
• RNA genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• Trypanosoma brucei brucei genetika   MeSH
• viabilita buněk genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• gBP21 protein, Trypanosoma brucei MeSH Prohlížeč
• mitochondriální proteiny MeSH
• proteiny vázající RNA MeSH
• protozoální proteiny MeSH
• RNA mitochondriální MeSH
• RNA MeSH

Histone acetylation modulates alternative splicing of several hundred genes. Here, we tested the role of the histone acetyltransferase p300 in alternative splicing and showed that knockdown of p300 promotes inclusion of the fibronectin (FN1) alternative EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as p300 knockdown. Next we showed that p300 controls histone H4 acetylation along the FN1 gene. Consistently, p300 depletion and CRE deletion/mutation both reduced histone H4 acetylation on mini-gene reporters. Finally, we provide evidence that the effect of CRE inactivation on H4 acetylation and alternative splicing is counteracted by the inhibition of histone deacetylases. Together, these data suggest that histone acetylation could be one of the mechanisms how promoter and promoter binding proteins influence alternative splicing.

• Klíčová slova
• alternative splicing, fibronectin, histone acetylation, p300, promoter,
• MeSH
• acetylace MeSH
• alternativní sestřih * MeSH
• fibronektiny genetika   metabolismus   MeSH
• genový knockdown MeSH
• HeLa buňky MeSH
• histony metabolismus   MeSH
• integrasy genetika   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• protein p300 asociovaný s E1A genetika   metabolismus   MeSH
• reportérové geny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Cre recombinase MeSH Prohlížeč
• EP300 protein, human MeSH Prohlížeč
• fibronektiny MeSH
• FN1 protein, human MeSH Prohlížeč
• histony MeSH
• integrasy MeSH
• messenger RNA MeSH
• protein p300 asociovaný s E1A MeSH

Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addition, almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunoprecipitation and live-cell imaging, we show that the C-terminal part is crucial for Brd2 association with chromatin. Live-cell microscopy also allows us to map the average binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.

• MeSH
• alternativní sestřih MeSH
• chromatin metabolismus   MeSH
• genetická transkripce MeSH
• genom lidský * MeSH
• HeLa buňky MeSH
• histony genetika   metabolismus   MeSH
• lidé MeSH
• promotorové oblasti (genetika) MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• signální transdukce MeSH
• terciární struktura proteinů MeSH
• transkripční faktory MeSH
• vazba proteinů MeSH
• videomikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BRD2 protein, human MeSH Prohlížeč
• chromatin MeSH
• histony MeSH
• protein-serin-threoninkinasy MeSH
• rekombinantní fúzní proteiny MeSH
• transkripční faktory MeSH