The ability of a cell to properly express its genes depends on optimal transcription and splicing. RNA polymerase II (RNAPII) transcribes protein-coding genes and produces pre-mRNAs, which undergo, largely co-transcriptionally, intron excision by the spliceosome complex. Spliceosome activation is a major control step, leading to a catalytically active complex. Recent work has showed that cyclin-dependent kinase (CDK)11 regulates spliceosome activation via the phosphorylation of SF3B1, a core spliceosome component. Thus, CDK11 arises as a major coordinator of gene expression in metazoans due to its role in the rate-limiting step of pre-mRNA splicing. This review outlines the evolution of CDK11 and SF3B1 and their emerging roles in splicing regulation. It also discusses how CDK11 and its inhibition affect transcription and cell cycle progression.

• Klíčová slova
• C-terminal domain of RNA polymerase II, OTS964, ULM–UHM interaction, cell cycle progression, constitutive splicing, histone transcription,
• MeSH
• buněčný cyklus MeSH
• cyklin-dependentní kinasy * metabolismus   genetika   MeSH
• lidé MeSH
• regulace genové exprese * MeSH
• sestřih RNA * genetika   MeSH
• sestřihové faktory MeSH
• spliceozomy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• cyklin-dependentní kinasy * MeSH
• sestřihové faktory MeSH

We have isolated and characterized a novel giant retroelement, named Ogre, which is over 22 kb long and makes up at least 5% of the pea (Pisum sativum L.) genome. This element can be classified as a Ty3/gypsy-like LTR retrotransposon based on the presence of long terminal repeats (LTRs) and the order of the domains coding for typical retrotransposon proteins. In addition to its extreme length, it has several features which make it unique among the retroelements described so far: (1) the sequences coding for gag and prot proteins are separated from the rt/rh-int domains by several stop codons; (2) the region containing these stop codons is removed from the element transcripts by splicing which results in reconstitution of the complete gag-pol coding sequence; (3) only a part of the transcripts is spliced which probably determines the ratio of translated proteins; (4) the element contains an extra ORF located upstream the gag-pol coding sequences, potentially coding for a protein of 546-562 amino acids with unknown function. The transcriptional activity of the Ogre elements has been detected in all organs tested (leaves, roots, flowers) as well as in wounded leaves and protoplasts. Considering this retroelement's constitutive expression and observed high mutual similarity of the element genomic sequences, it is possible to speculate about its recent amplification in the genomes of pea and other legume plants.

• MeSH
• alternativní sestřih * MeSH
• DNA rostlinná chemie   genetika   MeSH
• Fabaceae genetika   MeSH
• genetická transkripce * MeSH
• genom rostlinný MeSH
• hrách setý genetika   MeSH
• molekulární sekvence - údaje MeSH
• otevřené čtecí rámce genetika   MeSH
• regulace genové exprese u rostlin MeSH
• retroelementy genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie nukleových kyselin MeSH
• sekvenční seřazení MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• retroelementy MeSH

Introduction: Case-control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/-1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.

• Klíčová slova
• ACMG, RNAseq analysis, breast cancer, mRNA splicing, nanopore sequencing, variant classification,
• Publikační typ
• časopisecké články MeSH

Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3' splice site (3'ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3'ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3'ss distance and masked potential 3'ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3'ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3'ss recognition.

• MeSH
• Ascomycota genetika   MeSH
• fungální RNA chemie   MeSH
• introny * MeSH
• kofilin 1 genetika   MeSH
• konformace nukleové kyseliny MeSH
• místa sestřihu RNA * MeSH
• molekulární sekvence - údaje MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence nukleotidů MeSH
• sestřih RNA * MeSH
• teplota MeSH
• ubikvitin konjugující enzymy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• COF1 protein, S cerevisiae MeSH Prohlížeč
• fungální RNA MeSH
• kofilin 1 MeSH
• místa sestřihu RNA * MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• UBC13 protein, S cerevisiae MeSH Prohlížeč
• ubikvitin konjugující enzymy MeSH

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance.

• MeSH
• alternativní sestřih * MeSH
• Ascomycota imunologie   MeSH
• klonování DNA MeSH
• molekulární evoluce MeSH
• nemoci rostlin genetika   MeSH
• odolnost vůči nemocem genetika   MeSH
• proteinkinasy genetika   fyziologie   MeSH
• pšenice enzymologie   genetika   mikrobiologie   MeSH
• rekombinace genetická MeSH
• rostlinné geny MeSH
• rostlinné proteiny genetika   fyziologie   MeSH
• umlčování genů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteinkinasy MeSH
• rostlinné proteiny MeSH

In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

• MeSH
• alternativní sestřih genetika   MeSH
• chromatin metabolismus   MeSH
• exony genetika   MeSH
• fibronektiny genetika   MeSH
• genetická transkripce MeSH
• HeLa buňky MeSH
• histony metabolismus   MeSH
• lidé MeSH
• lysin metabolismus   MeSH
• metylace MeSH
• TAL efektory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• fibronektiny MeSH
• histony MeSH
• lysin MeSH
• TAL efektory MeSH

Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNAPhe, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote Trypanosoma brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNATyr in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.

• Klíčová slova
• Intron, queuosine, retrograde, splicing, tRNA, transport,
• MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro genetika   metabolismus   MeSH
• cytoplazma genetika   metabolismus   MeSH
• kinetika MeSH
• konformace nukleové kyseliny MeSH
• nukleosid Q metabolismus   MeSH
• pentosyltransferasy genetika   metabolismus   MeSH
• RNA transferová Phe genetika   metabolismus   MeSH
• RNA transferová Tyr genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sestřih RNA MeSH
• transport RNA MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• nukleosid Q MeSH
• pentosyltransferasy MeSH
• queuine tRNA-ribosyltransferase MeSH Prohlížeč
• RNA transferová Phe MeSH
• RNA transferová Tyr MeSH

BACKGROUND: An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments. METHODS: Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry. RESULTS: We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results. CONCLUSIONS: In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

• Klíčová slova
• TEEP-46, hypoxia, metabolism, mitochondria, pyruvate kinase, shikonin, splicing factors,
• MeSH
• alternativní sestřih MeSH
• antagomiry metabolismus   MeSH
• cévní endotel cytologie   MeSH
• fibroblasty cytologie   účinky léků   metabolismus   MeSH
• glykolýza MeSH
• heterogenní jaderné ribonukleoproteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• inhibitory histondeacetylas farmakologie   MeSH
• interleukin-1beta metabolismus   MeSH
• lidé MeSH
• makrofágy cytologie   imunologie   metabolismus   MeSH
• mikro RNA antagonisté a inhibitory   genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• naftochinony farmakologie   MeSH
• plicní hypertenze metabolismus   patologie   MeSH
• proliferace buněk MeSH
• protein - isoformy antagonisté a inhibitory   genetika   metabolismus   MeSH
• protein vázající polypyrimidinové úseky RNA antagonisté a inhibitory   genetika   metabolismus   MeSH
• pyruvátkinasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• RNA interference MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antagomiry MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• inhibitory histondeacetylas MeSH
• interleukin-1beta MeSH
• mikro RNA MeSH
• MIRN124 microRNA, human MeSH Prohlížeč
• naftochinony MeSH
• protein - isoformy MeSH
• protein vázající polypyrimidinové úseky RNA MeSH
• PTBP1 protein, human MeSH Prohlížeč
• pyruvátkinasa MeSH
• shikonin MeSH Prohlížeč

BACKGROUND: The programmed cell death 2 (Pdcd2) gene on mouse chromosome 17 was evaluated as a member of a highly conserved synteny, a candidate for an imprinted locus, and a candidate for the Hybrid sterility 1 (Hst1) gene. RESULTS: New mouse transcripts were identified at this locus: an alternative Pdcd2 mRNA skipping the last two coding exons and two classes of antisense RNAs. One class of the antisense RNA overlaps the alternative exon and the other the entire Pdcd2 gene. The antisense RNAs are alternative transcripts of the neighboring TATA-binding protein gene (Tbp) that are located mainly in the cell nucleus. Analogous alternative PDCD2 forms truncating the C-terminal domain were also detected in human and chicken. Alternative transcripts of the chicken PDCD2 and TBP genes also overlap. No correlation in the transcription of the alternative and overlapping mRNAs was detected. Allelic sequencing and transcription studies did not reveal any support for the candidacy of Pdcd2 for Hst1. No correlated expression of Pdcd2 with the other two genes of the highly conserved synteny was observed. Pdcd2, Chd1, and four other genes from this region were not imprinted in the embryo. CONCLUSION: The conservation of alternative transcription of the Pdcd2 gene in mouse, human and chicken suggests the biological importance of such truncated protein. The biological function of the alternative PDCD2 is likely to be opposite to that of the constitutive form. The ratio of the constitutive and alternative Pdcd2 mRNAs differs in the tissues, suggesting a developmental role. The identified Tbp-alternative Pdcd2-antisense transcripts may interfere with the transcription of the Pdcd2 gene, as they are transcribed at a comparable level. The conservation of the Pdcd2/Tbp sense-antisense overlap in the mouse and chicken points out its biological relevance. Our results also suggest that some cDNAs in databases labeled as noncoding are incomplete alternative cDNAs of neighboring protein-coding genes.

• MeSH
• alely MeSH
• alternativní sestřih * MeSH
• apoptóza genetika   MeSH
• exprimované sekvenční adresy MeSH
• genomový imprinting MeSH
• krysa rodu Rattus MeSH
• kur domácí MeSH
• lidé MeSH
• mapování chromozomů MeSH
• messenger RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• polymerázová řetězová reakce MeSH
• proteiny regulující apoptózu genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• Pdcd2 protein, mouse MeSH Prohlížeč
• proteiny regulující apoptózu MeSH

Calcium/calmodulin-dependent protein kinases (CaMKs) are key mediators of calcium signaling and underpin neuronal health. Although widely studied, the contribution of CaMKs to Mendelian disease is rather enigmatic. Here, we describe an unusual neurodevelopmental phenotype, characterized by milestone delay, intellectual disability, autism, ataxia, and mixed hyperkinetic movement disorder including severe generalized dystonia, in a proband who remained etiologically undiagnosed despite exhaustive testing. We performed trio whole-exome sequencing to identify a de novo essential splice-site variant (c.981+1G>A) in CAMK4, encoding CaMKIV. Through in silico evaluation and cDNA analyses, we demonstrated that c.981+1G>A alters CAMK4 pre-mRNA processing and results in a stable mRNA transcript containing a 77-nt out-of-frame deletion and a premature termination codon within the last exon. The expected protein, p.Lys303Serfs*28, exhibits selective loss of the carboxy-terminal regulatory domain of CaMKIV and bears striking structural resemblance to previously reported synthetic mutants that confer constitutive CaMKIV activity. Biochemical studies in proband-derived cells confirmed an activating effect of c.981+1G>A and indicated that variant-induced excessive CaMKIV signaling is sensitive to pharmacological manipulation. Additionally, we found that variants predicted to cause selective depletion of CaMKIV's regulatory domain are unobserved in diverse catalogs of human variation, thus revealing that c.981+1G>A is a unique molecular event. We propose that our proband's phenotype is explainable by a dominant CAMK4 splice-disrupting mutation that acts through a gain-of-function mechanism. Our findings highlight the importance of CAMK4 in human neurodevelopment, provide a foundation for future clinical research of CAMK4, and suggest the CaMKIV signaling pathway as a potential drug target in neurological disease.

• Klíčová slova
• athetoid cerebral palsy, language impairment, motor deterioration, psychomotor deterioration, torticollis,
• MeSH
• aktivační mutace genetika   MeSH
• cerebelární ataxie genetika   MeSH
• exom MeSH
• exony genetika   MeSH
• fenotyp MeSH
• hyperkineze genetika   MeSH
• lidé MeSH
• mentální retardace genetika   MeSH
• mutace MeSH
• nesmyslný kodon genetika   MeSH
• posunová mutace genetika   MeSH
• proteinkinasa závislá na vápníku a kalmodulinu typ 4 genetika   metabolismus   MeSH
• rodokmen MeSH
• sekvenování exomu MeSH
• sestřih RNA genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CAMK4 protein, human MeSH Prohlížeč
• nesmyslný kodon MeSH
• proteinkinasa závislá na vápníku a kalmodulinu typ 4 MeSH