Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.
- MeSH
- 5' nepřekládaná oblast MeSH
- eukaryotický iniciační faktor 2 genetika metabolismus MeSH
- eukaryotický iniciační faktor 3 genetika metabolismus MeSH
- HEK293 buňky MeSH
- iniciační faktory genetika metabolismus MeSH
- kodon iniciační MeSH
- lidé MeSH
- malé podjednotky ribozomu eukaryotické genetika metabolismus MeSH
- multiproteinové komplexy genetika metabolismus MeSH
- proteosyntéza * MeSH
- ribozomy genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika MeSH
- transkripční faktor ATF4 genetika metabolismus MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Reinitiation after translation of short upstream ORFs (uORFs) represents one of the means of regulation of gene expression on the mRNA-specific level in response to changing environmental conditions. Over the years it has been shown-mainly in budding yeast-that its efficiency depends on cis-acting features occurring in sequences flanking reinitiation-permissive uORFs, the nature of their coding sequences, as well as protein factors acting in trans. We earlier demonstrated that the first two uORFs from the reinitiation-regulated yeast GCN4 mRNA leader carry specific structural elements in their 5' sequences that interact with the translation initiation factor eIF3 to prevent full ribosomal recycling post their translation. Actually, this interaction turned out to be instrumental in stabilizing the mRNA·40S post-termination complex, which is thus capable to eventually resume scanning and reinitiate on the next AUG start site downstream. Recently, we also provided important in vivo evidence strongly supporting the long-standing idea that to stimulate reinitiation, eIF3 has to remain bound to ribosomes elongating these uORFs until their stop codon has been reached. Here we examined the importance of eIF3 and sequences flanking uORF1 of the human functional homolog of yeast GCN4, ATF4, in stimulation of efficient reinitiation. We revealed that the molecular basis of the reinitiation mechanism is conserved between yeasts and humans.
- MeSH
- eukaryotický iniciační faktor 3 chemie metabolismus MeSH
- iniciace translace peptidového řetězce * MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- otevřené čtecí rámce * MeSH
- proteosyntéza MeSH
- ribozomy metabolismus MeSH
- savci MeSH
- transkripční faktor ATF4 chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Tributyltin (TBT) is a ubiquitous contaminant in aquatic environment, but the detailed mechanisms underlying the toxicity of TBT have not been fully understood. In this study, the effects of TBT on behavior, energy metabolism and endoplasmic reticulum (ER) stress were investigated by using Chinese rare minnow larvae. Fish larvae were exposed at sublethal concentrations of TBT (100, 400 and 800 ng/L) for 7 days. Compared with the control, energy metabolic parameters (RNA/DNA ratio, Na(+)-K(+)-ATPase) were significantly inhibited in fish exposed at highest concentration (800 ng/L), as well as abnormal behaviors observed. Moreover, we found that the PERK (PKR-like ER kinase)-eIF2α (eukaryotic translation initiation factor 2α) pathway, as the main branch was activated by TBT exposure in fish larvae. In short, TBT-induced physiological, biochemical and molecular responses in fish larvae were reflected in parameters measured in this study, which suggest that these biomarkers could be used as potential indicators for monitoring organotin compounds present in aquatic environment.
- MeSH
- chování zvířat účinky léků MeSH
- Cyprinidae růst a vývoj MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- endoribonukleasy genetika metabolismus MeSH
- energetický metabolismus účinky léků MeSH
- kinasa eIF-2 genetika metabolismus MeSH
- larva účinky léků metabolismus MeSH
- stres endoplazmatického retikula účinky léků MeSH
- transkripční faktor ATF4 genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- trialkylcínové sloučeniny toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Čína MeSH