Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.
- MeSH
- exony * MeSH
- HeLa buňky MeSH
- lidé MeSH
- místa sestřihu RNA * MeSH
- RNA malá jaderná * metabolismus genetika MeSH
- sekvence nukleotidů MeSH
- sestřih RNA MeSH
- transkripční faktor STAT3 * metabolismus genetika MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.
- MeSH
- cytogenetické vyšetření metody MeSH
- genom MeSH
- hybridizace in situ fluorescenční MeSH
- mapování chromozomů MeSH
- molekulární evoluce * MeSH
- motýli genetika MeSH
- můry genetika MeSH
- ribozomální DNA genetika MeSH
- RNA malá jaderná genetika MeSH
- RNA ribozomální 18S genetika MeSH
- RNA ribozomální 5S genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.
- MeSH
- buněčné linie MeSH
- lidé MeSH
- mutace MeSH
- nádorový supresorový protein p53 biosyntéza genetika MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) MeSH
- proteiny genetika MeSH
- proteosyntéza * MeSH
- reportérové geny MeSH
- RNA malá jaderná genetika MeSH
- RNA transferová Trp genetika metabolismus MeSH
- RNA transferová Tyr genetika metabolismus MeSH
- terminační kodon * MeSH
- tryptofan-tRNA-ligasa genetika MeSH
- tyrosin-tRNA-ligasa genetika MeSH
- virové proteiny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.
- MeSH
- alternativní sestřih * MeSH
- buněčné linie MeSH
- exony * MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- místa sestřihu RNA MeSH
- mutace * MeSH
- mutageneze MeSH
- protein přežití motorických neuronů 1 chemie genetika metabolismus MeSH
- RNA malá jaderná chemie genetika metabolismus MeSH
- simulace molekulární dynamiky MeSH
- vazba proteinů MeSH
- výpočetní biologie metody MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.
- MeSH
- buněčné jádro genetika MeSH
- Cajalova tělíska genetika metabolismus MeSH
- HeLa buňky MeSH
- lidé MeSH
- malý jaderný ribonukleoprotein U2 genetika MeSH
- regulace genové exprese genetika MeSH
- RNA malá jaderná genetika MeSH
- spliceozomy genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
