tRNA modification Dotaz Zobrazit nápovědu
Organisms have evolved different strategies to seclude certain molecules to specific locations of the cell. This is most pronounced in eukaryotes with their extensive intracellular membrane systems. Intracellular compartmentalization is particularly critical in genome containing organelles, which because of their bacterial evolutionary ancestry still maintain protein-synthesis machinery that resembles more their evolutionary origin than the extant eukaryotic cell they once joined as an endosymbiont. Despite this, it is clear that genome-containing organelles such as the mitochondria are not in isolation and many molecules make it across the mitochondrial membranes from the cytoplasm. In this realm the import of tRNAs and the enzymes that modify them prove most consequential. In this review, we discuss two recent examples of how modifications typically found in cytoplasmic tRNAs affect mitochondrial translation in organisms that forcibly import all their tRNAs from the cytoplasm. In our view, the combination of tRNA import and the compartmentalization of modification enzymes must have played a critical role in the evolution of the organelle. © 2018 IUBMB Life, 70(12):1207-1213, 2018.
- MeSH
- cytoplazma genetika MeSH
- genom mitochondriální genetika MeSH
- intracelulární membrány MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondrie genetika MeSH
- posttranskripční úpravy RNA genetika MeSH
- proteosyntéza genetika MeSH
- RNA transferová genetika MeSH
- symbióza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.
- MeSH
- guanosin analogy a deriváty biosyntéza chemie metabolismus MeSH
- mitochondrie enzymologie MeSH
- posttranskripční úpravy RNA MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA transferová chemie metabolismus MeSH
- Trypanosoma brucei brucei enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in β cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t6A37 in tRNALysUUU to ms2t6A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms2t6A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2-/- β cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.
- MeSH
- beta-buňky metabolismus MeSH
- homeostáza MeSH
- inzulin metabolismus MeSH
- inzulinom genetika metabolismus MeSH
- krysa rodu rattus MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- nádorové buněčné linie MeSH
- nádory slinivky břišní genetika metabolismus MeSH
- porucha glukózové tolerance genetika MeSH
- proinsulin genetika metabolismus MeSH
- protein 2 regulující obsah železa genetika metabolismus MeSH
- proteiny obsahující železo a síru metabolismus MeSH
- RNA transferová Lys genetika metabolismus MeSH
- signální dráha UPR genetika MeSH
- tRNA-methyltransferasy genetika metabolismus MeSH
- železo metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Human cytoplasmic tRNAs contain dihydrouridine modifications at positions 16 and 17 (D16/D17). The enzyme responsible for D16/D17 formation and its cellular roles remain elusive. Here, we identify DUS1L as the human tRNA D16/D17 writer. DUS1L knockout in the glioblastoma cell lines LNZ308 and U87 causes loss of D16/D17. D formation is reconstituted in vitro using recombinant DUS1L in the presence of NADPH or NADH. DUS1L knockout/overexpression in LNZ308 cells shows that DUS1L supports cell growth. Moreover, higher DUS1L expression in glioma patients is associated with poorer prognosis. Upon vector-mediated DUS1L overexpression in LNZ308 cells, 5' and 3' processing of precursor tRNATyr(GUA) is inhibited, resulting in a reduced mature tRNATyr(GUA) level, reduced translation of the tyrosine codons UAC and UAU, and reduced translational readthrough of the near-cognate stop codons UAA and UAG. Moreover, DUS1L overexpression increases the amounts of several D16/D17-containing tRNAs and total cellular translation. Our study identifies a human dihydrouridine writer, providing the foundation to study its roles in health and disease.
- MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proteosyntéza * MeSH
- RNA transferová * metabolismus genetika MeSH
- uridin metabolismus analogy a deriváty MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Transfer RNAs play a key role in protein synthesis. Following transcription, tRNAs are extensively processed prior to their departure from the nucleus to become fully functional during translation. This includes removal of 5′ leaders and 3′ trailers by a specific endo- and/or exonuclease, 3′ CCA tail addition, posttranscriptional modifications and in some cases intron removal. In this minireview, the critical factors of nuclear tRNA trafficking are described based on studies in classical models such as yeast and human cell lines. In addition, recent findings and identification of novel regulatory loops of nuclear tRNA trafficking in trypanosomes are discussed with emphasis on tRNA modifications. The comparison between the representatives of opisthokonts and excavates serves here to understand the evolutionary conservation and diversity of nuclear tRNA export mechanisms.
- MeSH
- buněčné linie MeSH
- lidé MeSH
- RNA jaderná genetika metabolismus MeSH
- RNA transferová genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Trypanosoma genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Transfer RNA isopentenylation an adenine 37 position (A37) is a universal modification known in prokaryotes and eukaryotes. A set of highly homologous enzymes catalyse a series of reactions, leading to tRNA modifications, aimed to increase adaptation to environmental condition through the control of translation efficiency and reading frame maintenance. Transfer RNA-isopentenylation-related (TI-related) functions are well studied in bacteria, mitochondria of yeast and human, but completely unexplored in plants. Transfer RNA-isopentenylation-unrelated (TI-unrelated) functions participate in adaptation to environmental stresses via the regulation of sterol metabolism, gene silencing/suppression and amyloid fibrils formation. TI-unrelated functions are mostly studied in yeast. Finally, the degradation of A37-modified tRNA releases a set of bioactive compounds known as cis-cytokinins. Although all organisms are able to produce cis-cytokinins, its physiological role is still a matter of debates. For several species of bacteria and fungi, cis-cytokinins are known to play a crucial role in pathogenesis. In mammalian and human models cis-cytokinins have tumour-suppressing and anti-inflammation effects. This review aims to summarise current knowledge of the TI-related and TI-unrelated functions and main bioactive by-products of isopentenylated tRNA degradation.
Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.
- MeSH
- adenosintrifosfát metabolismus MeSH
- aminoacyl-tRNA-synthetasy antagonisté a inhibitory MeSH
- biotest metody MeSH
- inhibitory enzymů chemie izolace a purifikace farmakologie MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- ligandy MeSH
- molekulární modely MeSH
- počítačová simulace * MeSH
- pyrazinamid chemie MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.
- MeSH
- bakteriální toxiny genetika metabolismus farmakologie MeSH
- fosforylace účinky léků MeSH
- grampozitivní nesporulující tyčinky chemie metabolismus MeSH
- guanosinpentafosfát chemie metabolismus MeSH
- inhibitory syntézy proteinů farmakologie MeSH
- ligasy chemie genetika metabolismus MeSH
- proteosyntéza účinky léků fyziologie MeSH
- pyrofosfatasy MeSH
- ribozomy metabolismus MeSH
- RNA transferová metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Fe/S clusters are part of the active site of many enzymes and are essential for cell viability. In eukaryotes the cysteine desulfurase Nfs (IscS) donates the sulfur during Fe/S cluster assembly and was thought sufficient for this reaction. Moreover, Nfs is indispensable for tRNA thiolation, a modification generally required for tRNA function and protein synthesis. Recently, Isd11 was discovered as an integral part of the Nfs activity at an early step of Fe/S cluster assembly. Here we show, using a combination of genetic, molecular, and biochemical approaches, that Isd11, in line with its strong association with Nfs, is localized in the mitochondrion of T. brucei. In addition to its involvement in Fe/S assembly, Isd11 also partakes in both cytoplasmic and mitochondrial tRNA thiolation, whereas Mtu1, another protein proposed to collaborate with Nfs in tRNA thiolation, is required for this process solely within the mitochondrion. Taken together these data place Isd11 at the center of these sulfur transactions and raises the possibility of a connection between Fe/S metabolism and protein synthesis, helping integrate two seemingly unrelated pathways.
- MeSH
- akonitáthydratasa metabolismus MeSH
- cytosol metabolismus MeSH
- fenotyp MeSH
- fumarasa metabolismus MeSH
- membránový potenciál mitochondrií MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- proteiny obsahující železo a síru metabolismus MeSH
- protozoální proteiny metabolismus MeSH
- RNA interference MeSH
- RNA protozoální metabolismus MeSH
- RNA transferová metabolismus MeSH
- stabilita proteinů MeSH
- sulfhydrylové sloučeniny metabolismus MeSH
- Trypanosoma brucei brucei cytologie enzymologie růst a vývoj 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
Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U-containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U-containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.
