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

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.

• Keywords
• H2A.Z, Synthetic genetic array analysis, chromatin modifiers, co-transcriptional splicing, spliceosome assembly,
• MeSH
• Phenotype * MeSH
• Histones metabolism   genetics   MeSH
• Introns * MeSH
• Gene Expression Regulation, Fungal MeSH
• Saccharomyces cerevisiae Proteins * genetics   metabolism   MeSH
• Saccharomyces cerevisiae * genetics   metabolism   MeSH
• RNA Splicing * MeSH
• RNA Splicing Factors genetics   metabolism   MeSH
• Spliceosomes * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Histones MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• RNA Splicing Factors 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.

• Keywords
• immunoglobulin E, lipid raft, mast cell, plasma membrane, signal transduction, tumor necrosis factor, tyrosine kinase,
• MeSH
• Antigens metabolism   MeSH
• Cell Degranulation MeSH
• Ursolic Acid MeSH
• Lipids pharmacology   MeSH
• Mast Cells metabolism   MeSH
• Mice MeSH
• Receptors, IgE * metabolism   MeSH
• Triterpenes * pharmacology   metabolism   MeSH
• Calcium metabolism   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
• Antigens MeSH
• Lipids MeSH
• Receptors, IgE * MeSH
• Triterpenes * MeSH
• Calcium 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

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.

• Keywords
• BBSome, Bardet-Biedl Syndrome, Bardet-Biedl syndrome, assembly, ciliopathy, cilium, genetic disease, microscopic imaging, primary cilium, protein assembly, protein sorting,
• MeSH
• Bardet-Biedl Syndrome genetics   metabolism   pathology   MeSH
• Cell Line MeSH
• Cilia metabolism   MeSH
• CRISPR-Cas Systems genetics   MeSH
• Cytoplasm metabolism   MeSH
• Gene Editing MeSH
• Microscopy, Fluorescence MeSH
• Fluorescence Recovery After Photobleaching MeSH
• Humans MeSH
• Mutation MeSH
• Protein Subunits genetics   metabolism   MeSH
• Microtubule-Associated Proteins deficiency   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bbs1 protein, human MeSH Browser
• BBS4 protein, human MeSH Browser
• Protein Subunits MeSH
• Microtubule-Associated Proteins 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
• Amino Acid Motifs MeSH
• Exons * MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Heterogeneous-Nuclear Ribonucleoproteins metabolism   MeSH
• Nuclear Proteins metabolism   MeSH
• Short Interspersed Nucleotide Elements MeSH
• Humans MeSH
• Ribonucleoprotein, U1 Small Nuclear metabolism   MeSH
• Mutation, Missense * MeSH
• RNA Splice Sites * MeSH
• RNA-Binding Proteins metabolism   MeSH
• Repressor Proteins chemistry   deficiency   metabolism   MeSH
• Sequence Analysis, RNA MeSH
• RNA Splicing Factors chemistry   deficiency   metabolism   MeSH
• Splicing Factor U2AF MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• HCC1 autoantigen MeSH Browser
• Heterogeneous-Nuclear Ribonucleoproteins MeSH
• Nuclear Proteins MeSH
• Ribonucleoprotein, U1 Small Nuclear MeSH
• RNA Splice Sites * MeSH
• poly-U binding splicing factor 60KDa MeSH Browser
• RNA-Binding Proteins MeSH
• Repressor Proteins MeSH
• RNA Splicing Factors MeSH
• Splicing Factor U2AF MeSH
• SNRNP70 protein, human MeSH Browser

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.

• Keywords
• Retinitis pigmentosa, snRNP, splicing,
• MeSH
• Introns MeSH
• Rats MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Mice MeSH
• RNA Precursors genetics   metabolism   MeSH
• Retinitis Pigmentosa genetics   MeSH
• Ribonucleoproteins, Small Nuclear genetics   metabolism   MeSH
• RNA Splicing MeSH
• RNA Splicing Factors genetics   metabolism   MeSH
• Spliceosomes genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA Precursors MeSH
• Ribonucleoproteins, Small Nuclear MeSH
• RNA Splicing Factors 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
• Mitochondrial Proteins genetics   MeSH
• Mutation MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• RNA Interference MeSH
• RNA, Mitochondrial MeSH
• RNA genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Trypanosoma brucei brucei genetics   MeSH
• Cell Survival genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• gBP21 protein, Trypanosoma brucei MeSH Browser
• Mitochondrial Proteins MeSH
• RNA-Binding Proteins MeSH
• Protozoan Proteins MeSH
• RNA, Mitochondrial 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.

• Keywords
• alternative splicing, fibronectin, histone acetylation, p300, promoter,
• MeSH
• Acetylation MeSH
• Alternative Splicing * MeSH
• Fibronectins genetics   metabolism   MeSH
• Gene Knockdown Techniques MeSH
• HeLa Cells MeSH
• Histones metabolism   MeSH
• Integrases genetics   MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Promoter Regions, Genetic MeSH
• E1A-Associated p300 Protein genetics   metabolism   MeSH
• Genes, Reporter MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cre recombinase MeSH Browser
• EP300 protein, human MeSH Browser
• Fibronectins MeSH
• FN1 protein, human MeSH Browser
• Histones MeSH
• Integrases MeSH
• RNA, Messenger MeSH
• E1A-Associated p300 Protein 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
• Alternative Splicing MeSH
• Chromatin metabolism   MeSH
• Transcription, Genetic MeSH
• Genome, Human * MeSH
• HeLa Cells MeSH
• Histones genetics   metabolism   MeSH
• Humans MeSH
• Promoter Regions, Genetic MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Gene Expression Regulation * MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Signal Transduction MeSH
• Protein Structure, Tertiary MeSH
• Transcription Factors MeSH
• Protein Binding MeSH
• Microscopy, Video MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BRD2 protein, human MeSH Browser
• Chromatin MeSH
• Histones MeSH
• Protein Serine-Threonine Kinases MeSH
• Recombinant Fusion Proteins MeSH
• Transcription Factors MeSH