RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

• MeSH
• Enzyme Activation drug effects   MeSH
• Quinolones pharmacology   MeSH
• Chromatin metabolism   MeSH
• Cyclin-Dependent Kinases * antagonists & inhibitors   metabolism   MeSH
• Phosphoproteins * chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Ribonucleoprotein, U2 Small Nuclear * chemistry   metabolism   MeSH
• RNA Precursors * genetics   metabolism   MeSH
• RNA Splicing * drug effects   MeSH
• Spliceosomes * drug effects   metabolism   MeSH
• Threonine metabolism   MeSH
• Publication type
• Journal Article MeSH

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

• MeSH
• Cell Nucleus metabolism   MeSH
• Cell Line MeSH
• Spectrometry, Fluorescence MeSH
• Fluorescence Recovery After Photobleaching MeSH
• HeLa Cells MeSH
• Kinetics MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• RNA Precursors metabolism   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   physiology   MeSH
• RNA Splicing physiology   MeSH
• Spliceosomes metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1) strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok) pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd) can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

• MeSH
• Cell Cycle genetics   MeSH
• Drosophila melanogaster genetics   MeSH
• Ecdysone genetics   MeSH
• Cells, Cultured MeSH
• Larva genetics   MeSH
• Mutation genetics   MeSH
• RNA Precursors genetics   MeSH
• Drosophila Proteins genetics   MeSH
• Ribonucleoproteins, Small Nuclear genetics   MeSH
• RNA Splicing genetics   MeSH
• Spliceosomes genetics   MeSH
• Steroids metabolism   MeSH
• Gene Expression Regulation, Developmental genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Models, Biological MeSH
• Cell Nucleus metabolism   MeSH
• Molecular Structure MeSH
• RNA Precursors genetics   chemistry   metabolism   MeSH
• RNA Splicing MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Review MeSH

UBL5 is an atypical ubiquitin-like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre-mRNA splicing efficiency, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre-mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions, our results show that UBL5 plays an evolutionary conserved role in pre-mRNA splicing, the integrity of which is essential for the fidelity of chromosome segregation.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   MeSH
• Chromatids genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ligases genetics   MeSH
• Mitosis genetics   MeSH
• Eye Proteins genetics   metabolism   MeSH
• RNA Precursors genetics   MeSH
• Cell Cycle Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• Saccharomyces cerevisiae Proteins genetics   MeSH
• Chromosome Segregation genetics   MeSH
• RNA Splicing genetics   MeSH
• Spliceosomes genetics   MeSH
• Ubiquitins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Familial dysautonomia is a debilitating congenital neurodegenerative disorder with no causative therapy. It is caused by a homozygous mutation in ELP1 gene, resulting in the production of the transcript lacking exon 20. The compounds studied as potential treatments include the clinical candidate kinetin, a plant hormone from the cytokinin family. We explored the relationship between the structure of a set of kinetin derivatives (N = 72) and their ability to correct aberrant splicing of the ELP1 gene. Active compounds can be obtained by the substitution of the purine ring with chlorine and fluorine at the C2 atom, with a small alkyl group at the N7 atom, or with diverse groups at the C8 atom. On the other hand, a substitution at the N3 or N9 atoms resulted in a loss of activity. We successfully tested a hypothesis inspired by the remarkable tolerance of the position C8 to substitution, postulating that the imidazole of the purine moiety is not required for the activity. We also evaluated the activity of phytohormones from other families, but none of them corrected ELP1 mRNA aberrant splicing. A panel of in vitro ADME assays, including evaluation of transport across model barriers, stability in plasma and in the presence of liver microsomal fraction as well as plasma protein binding, was used for an initial estimation of the potential bioavailability of the active compounds. Finally, a RNA-seq data suggest that 8-aminokinetin modulates expression spliceosome components.

• MeSH
• Kinetin * pharmacology   chemistry   MeSH
• Humans MeSH
• Molecular Structure MeSH
• RNA Precursors * genetics   metabolism   MeSH
• RNA Splicing * drug effects   MeSH
• Transcriptional Elongation Factors metabolism   genetics   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Prevence vzniku nádorů u nosičů dědičných alterací nádorových predispozičních genů vyžaduje jasnou identifikaci zodpovědných patogenních mutací. Při analýze nádorové predispozice je odhalena řada variant s nejasným významem (VUS) komplikujících klinickou interpretaci výsledků. Pokud VUS postihují proces sestřihu transkriptu příslušného genu, lze je považovat za patogenní. V projektu se zaměříme na vyhodnocení změn sestřihového vzorce mRNA transkriptů u nosičů dědičných variant genů predisponujících ke vzniku dědičné formy karcinomu (ca) prsu a ovarií (CHEK2, PALB2, NBN, TP53, RAD51C/D, MLH1, MSH2/6, BRIP1). Nejprve provedeme identifikaci jejich sestřihových variant v normálních tkáních (leukocyty, mamární a tuková tkáň) zdravých osob bez přítomnosti dědičných variant pomocí multiplexní PCR (mPCR) umožňující amplifikaci všech sestřihových oblastí s následnou charakterizací sekvenovánín nové generace (NGS). Vyšetření RNA od nosičů VUS umožní kvalitativně (fragmentační a mPCR/NGS analýza) i kvantitativně (qPCR) identifikovat VUS způsobující změny sestřihového vzorce příslušného genu.; Clinical management that enables efficient cancer prevention in carriers of germ-line alterations in cancer susceptibility genes requires and unequivocal characterization of the pathogenic mutation. Current cancer predisposition analyses reveal many variants of unknown clinical significance (VUS) which complicate the clinical interpretation of results. The VUS that change splicing of corresponding gene product may be considered as pathogenic. Our project aims to identify splicing alterations in carriers of VUS in genes predisposing to breast and ovarian cancer (CHEK2, PALB2, NBN, TP53, RAD51C/D, MLH1, MSH2/6, BRIP1). First, we aim to perform a comprehensive characterization of naturally occurring splicing variants of analyzed genes in normal lymphocytes, mammary and adipose tissues from non-cancer individuals using multiplex (m)PCR/next generation sequencing (NGS) analysis. Subsequently, the analyses of RNA from carriers of VUS will enable to characterize the qualitative (fragment analysis, mPCR/NGS) and quantitative (qPCR) changes in splicing pattern of corresponding genes.

• MeSH
• Hereditary Breast and Ovarian Cancer Syndrome genetics   classification   MeSH
• Genetic Predisposition to Disease genetics   classification   MeSH
• Genes, Neoplasm genetics   MeSH
• RNA Precursors MeSH
• RNA Splicing genetics   MeSH
• RNA Splicing Factors MeSH
• High-Throughput Nucleotide Sequencing MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• onkologie
• genetika, lékařská genetika
• gynekologie a porodnictví
• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• biochemie
• mikrobiologie, lékařská mikrobiologie

There are numerous data suggesting that two key steps in gene expression-transcription and splicing influence each other closely. For a long time it was known that chromatin modifications regulate transcription, but only recently it was shown that chromatin and histone modifications play a significant role in pre-mRNA splicing. Here we summarize interactions between splicing machinery and chromatin and discuss their potential functional significance. We focus mainly on histone acetylation and methylation and potential mechanisms of their role in splicing. It seems that whereas histone acetylation acts mainly by alterating the transcription rate, histone methylation can also influence splicing directly by recruiting various splicing components.

• MeSH
• Chromatin genetics   metabolism   MeSH
• Transcription, Genetic genetics   MeSH
• Humans MeSH
• Nucleoproteins metabolism   MeSH
• RNA Precursors genetics   MeSH
• RNA Splicing MeSH
• Transcription Factors metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• alergologie a imunologie
• mikrobiologie, lékařská mikrobiologie