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.

• Klíčová slova
• UBL5, pre‐mRNA splicing, sister chromatid cohesion, sororin, ubiquitin‐like protein,
• MeSH
• adaptorové proteiny signální transdukční genetika   MeSH
• chromatidy genetika   MeSH
• HeLa buňky MeSH
• lidé MeSH
• ligasy genetika   MeSH
• mitóza genetika   MeSH
• oční proteiny genetika   metabolismus   MeSH
• prekurzory RNA genetika   MeSH
• proteiny buněčného cyklu genetika   MeSH
• regulace genové exprese MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• segregace chromozomů genetika   MeSH
• sestřih RNA genetika   MeSH
• spliceozomy genetika   MeSH
• ubikvitiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• CDCA5 protein, human MeSH Prohlížeč
• HUB1 protein, S cerevisiae MeSH Prohlížeč
• ligasy MeSH
• oční proteiny MeSH
• prekurzory RNA MeSH
• proteiny buněčného cyklu MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• ubikvitiny MeSH
• UBL5 protein, human MeSH Prohlížeč

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
• aktivace enzymů účinky léků   MeSH
• chinolony farmakologie   MeSH
• chromatin metabolismus   MeSH
• cyklin-dependentní kinasy * antagonisté a inhibitory   metabolismus   MeSH
• fosfoproteiny * chemie   metabolismus   MeSH
• fosforylace MeSH
• malý jaderný ribonukleoprotein U2 * chemie   metabolismus   MeSH
• prekurzory RNA * genetika   metabolismus   MeSH
• sestřih RNA * účinky léků   MeSH
• spliceozomy * účinky léků   metabolismus   MeSH
• threonin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chinolony MeSH
• chromatin MeSH
• cyklin-dependentní kinasy * MeSH
• fosfoproteiny * MeSH
• malý jaderný ribonukleoprotein U2 * MeSH
• OTS964 MeSH Prohlížeč
• prekurzory RNA * MeSH
• threonin MeSH

Several models have been presented in the past to explain localized distributions of nuclear RNAs from individual genes that range from small foci to more elongated "track-like" structures. We present here a hypothesis which explains that, in the case of regulated splicing, there is in diploid cells a spatial separation of transcription sites from the execution of regulated splicing which we situate to domains of SR protein accumulation. In addition, it explains the presence of poly(A) sequences, and the lack of the autoradiographic label due to short pulses of [3H]uridine, in these domains.

• MeSH
• biologické modely MeSH
• buněčné jádro metabolismus   MeSH
• molekulární struktura MeSH
• prekurzory RNA chemie   genetika   metabolismus   MeSH
• sestřih RNA * MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• prekurzory RNA 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
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• fluorescenční spektrometrie MeSH
• FRAP MeSH
• HeLa buňky MeSH
• kinetika MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• prekurzory RNA metabolismus   MeSH
• ribonukleoproteiny malé jaderné metabolismus   fyziologie   MeSH
• sestřih RNA fyziologie   MeSH
• spliceozomy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• prekurzory RNA MeSH
• ribonukleoproteiny malé jaderné 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
• buněčný cyklus genetika   MeSH
• Drosophila melanogaster genetika   MeSH
• ekdyson genetika   MeSH
• kultivované buňky MeSH
• larva genetika   MeSH
• mutace genetika   MeSH
• prekurzory RNA genetika   MeSH
• proteiny Drosophily genetika   MeSH
• ribonukleoproteiny malé jaderné genetika   MeSH
• sestřih RNA genetika   MeSH
• spliceozomy genetika   MeSH
• steroidy metabolismus   MeSH
• vývojová regulace genové exprese genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ecd protein, Drosophila MeSH Prohlížeč
• ekdyson MeSH
• prekurzory RNA MeSH
• proteiny Drosophily MeSH
• ribonukleoproteiny malé jaderné MeSH
• steroidy MeSH

Förster resonance energy transfer (FRET) microscopy is a powerful technique routinely used to monitor interactions between biomolecules. Here, we focus on the techniques that are used for investigating the structure and interactions of nucleic acids (NAs). We present a brief overview of the most commonly used FRET microscopy techniques, their advantages and drawbacks. We list experimental approaches recently used for either in vitro or in vivo studies. Next, we summarize how FRET contributed to the understanding of pre-mRNA splicing and spliceosome assembly.

• MeSH
• alternativní sestřih MeSH
• fluorescenční mikroskopie * MeSH
• molekulární konformace MeSH
• nukleoproteiny metabolismus   MeSH
• nukleové kyseliny chemie   genetika   metabolismus   MeSH
• prekurzory RNA chemie   genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie * MeSH
• sestřih RNA * MeSH
• spliceozomy genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• nukleoproteiny MeSH
• nukleové kyseliny MeSH
• prekurzory RNA MeSH

For more than three decades, researchers have known that consensus splice sites alone are not sufficient regulatory elements to provide complex splicing regulation. Other regulators, so-called splicing regulatory elements (SREs) are needed. Most importantly, their sequence variants often underlie the development of various human disorders. However, due to their variable location and high degeneracy, these regulatory sequences are also very difficult to recognize and predict. Many different approaches aiming to identify SREs have been tried, often leading to the development of in silico prediction tools. While these tools were initially expected to be helpful to identify splicing-affecting mutations in genetic diagnostics, we are still quite far from meeting this goal. In fact, most of these tools are not able to accurately discern the SRE-affecting pathological variants from those not affecting splicing. Nonetheless, several recent evaluations have given appealing results (namely for EX-SKIP, ESRseq and Hexplorer predictors). In this review, we aim to summarize the history of the different approaches to SRE prediction, and provide additional validation of these tools based on patients' clinical data. Finally, we evaluate their usefulness for diagnostic settings and discuss the challenges that have yet to be met.

• Klíčová slova
• evaluation of prediction tools, in silico predictions, mutation, pre-mRNA splicing, splicing aberration, splicing regulatory elements, variants of unknown significance,
• MeSH
• diagnostické techniky molekulární metody   trendy   MeSH
• genetické nemoci vrozené * MeSH
• lidé MeSH
• místa sestřihu RNA * MeSH
• mutace * MeSH
• prekurzory RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• místa sestřihu RNA * MeSH
• prekurzory RNA MeSH

Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5' and 3' splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.

• MeSH
• databáze nukleových kyselin MeSH
• mutace genetika   MeSH
• prekurzory RNA genetika   MeSH
• průběh práce * MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvenční analýza RNA metody   MeSH
• sestřih RNA genetika   MeSH
• spliceozomy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• prekurzory RNA 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.

• Klíčová slova
• ADME in vitro, Alternative splicing, Cytokinin, ELP1, Kinetin, mRNA metabolism,
• MeSH
• kinetin * farmakologie   chemie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• prekurzory RNA * genetika   metabolismus   MeSH
• sestřih RNA * účinky léků   MeSH
• transkripční elongační faktory metabolismus   genetika   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Elp1 protein, human MeSH Prohlížeč
• kinetin * MeSH
• prekurzory RNA * MeSH
• transkripční elongační faktory MeSH

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein-Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.

• MeSH
• biologický transport MeSH
• buněčné jádro genetika   metabolismus   ultrastruktura   virologie   MeSH
• DNA řízené RNA-polymerasy antagonisté a inhibitory   metabolismus   MeSH
• DNA virů genetika   metabolismus   MeSH
• elektronová mikroskopie MeSH
• fluorescenční mikroskopie MeSH
• genetická transkripce genetika   MeSH
• genom virový MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• introny genetika   MeSH
• jaderné proteiny metabolismus   MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nádorové buňky kultivované MeSH
• plazmidy genetika   MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• ribonukleoproteiny metabolismus   MeSH
• RNA virová genetika   metabolismus   MeSH
• serin-arginin sestřihové faktory MeSH
• spliceozomy genetika   metabolismus   ultrastruktura   MeSH
• virové geny genetika   MeSH
• virus Epsteinův-Barrové genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• DNA virů MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• jaderné proteiny MeSH
• messenger RNA MeSH
• prekurzory RNA MeSH
• ribonukleoproteiny MeSH
• RNA virová MeSH
• serin-arginin sestřihové faktory MeSH
• SRSF2 protein, human MeSH Prohlížeč