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Introduction Mutations in QARS1, which encodes human glutaminyl-tRNA synthetase, have been associated with epilepsy, developmental regression, progressive microcephaly and cerebral atrophy. Epilepsy caused by variants in QARS1 is usually drug-resistant and intractable. Childhood onset epilepsy is also reported in various aminoacyl-tRNA synthetase disorders. We describe a case with a milder neurological phenotype than previously reported with QARS1 variants and review the seizure associations with aminoacyl-tRNA synthetase disorders. Case report The patient is a 4-year-old girl presenting at 6 weeks of age with orofacial dyskinesia and hand stereotypies. She developed focal seizures at 7 months of age. Serial electroencephalograms showed shifting focality. Her seizures were controlled after introduction of carbamazepine. Progress MRI showed very mild cortical volume loss without myelination abnormalities or cerebellar atrophy. She was found to have novel compound heterozygous variants in QARS1 (NM_005051.2): c.[1132C > T];[1574G > A], p.[(Arg378Cys)];[(Arg525Gln)] originally classified as “variants of uncertain significance” and later upgraded to “likely pathogenic” based on functional testing and updated variant database review. Functional testing showed reduced solubility of the corresponding QARS1 mutants in vitro, but only mild two-fold loss in catalytic efficiency with the c.1132C > T variant and no noted change in tRNAGln aminoacylation with the c.1574G > A variant. Conclusion We describe two QARS1 variants associated with overall conserved tRNA aminoacylation activity but characterized by significantly reduced QARS protein solubility, resulting in a milder clinical phenotype. 86% of previous patients reported with QARS1 had epilepsy and 79% were pharmaco-resistant. We also summarise literature regarding epilepsy in aminoacyl-tRNA synthetase disorders, which is also often early onset, severe and drug-refractory.

Pigeon milk is a curdlike substance separated from the mature crop epithelium of breeders, associated with the rapid growth and development of squabs. The aim of this study was to investigate in detail the variations in the content of several important ingredients in crop milk. In this study, we utilized proteomic techniques to investigate the composition and changing pattern of crop milk protein of squabs on days 1 (D1), 3 (D3), and 7 (D7). Our results indicated that the crude protein contents in crop milk decreased with age, and they were up to 50% during the first 3 days. The proteomic data showed that a total of 2558 proteins were identified in all samples from three stages, and the top 15% crop milk proteins were ribosomal protein, keratin, peroxiredoxin, annexin, heat shock protein, and eukaryotic translation protein based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and normalized spectral abundance factors (NSAFs) calculation. Furthermore, the compositions of crop milk protein between D1 and D3 were quite similar [51 differentially expressed proteins (DEPs)], while great proteomic differences were observed between D1/D3 and D7 (more than 240 DEPs). Additionally, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that up-regulated DEPs mainly participate in immune response, while down-regulated DEPs were involved in cell differentiation and development as well as tRNA aminoacylation biosynthesis. In conclusion, DEPs were mainly related to protein synthesis, immunity, and antioxidation, which provided effective information for the development of artificial squab milk products in the future.

Summary Protein translation from the ribosome seemingly requires dozens of sophisticated aminoacyl-tRNA synthetases (aaRSs). Despite the discovery of tRNA-aminoacylating ribozymes, such as the flexizyme, synthesizing protein enzymes from highly simplified translation systems in the absence of aaRS remains undemonstrated. Here, we show the translation of multiple protein enzymes of distinct functions using solely flexizyme-charged tRNAs, through improving the translation yield by concentrating cation-depleted tRNAs. Notably, we used the aaRS-free translation system to produce an active aaRS (TrpRS), which in turn catalyzed the charging of more tRNAs. Moreover, toward realizing mirror-image translation, we performed the mirror-image tRNA charging with D-amino acids by a synthetic L-flexizyme. Our work demonstrates the feasibility of translating protein enzymes from a highly simplified translation apparatus without aaRS and drastically reduces the requirement to chemically synthesize dozens of large aaRS proteins for realizing mirror-image translation.

Background Cross talk of tumor–immune cells at the gene expression level has been an area of intense research. However, it is largely unknown at the alternative splicing level which has been found to play important roles in the tumor–immune microenvironment. Results Here, we re-exploited one transcriptomic dataset to gain insight into tumor–immune interactions from the point of AS level. Our results showed that the AS profiles of triple-negative breast cancer cells co-cultured with activated T cells were significantly changed but not Estrogen receptor positive cells. We further suggested that the alteration in AS profiles in triple-negative breast cancer cells was largely caused by activated T cells rather than paracrine factors from activated T cells. Biological pathway analyses showed that translation initiation and tRNA aminoacylation pathways were most disturbed with T cell treatment. We also established an approach largely based on the AS factor–AS events associations and identified LSM7, an alternative splicing factor, may be responsible for the major altered events. Conclusions Our study reveals the notable differences of response to T cells among breast cancer types which may facilitate the development or improvement of tumor immunotherapy. How to cite: Zhao L, Yang X, Feng C, et al. Triple-negative breast cancer cells respond to T cells severely at the alternative splicing layer. Electron J Biotechnol 2021;50. https://doi.org/10.1016/j.ejbt.2021.01.001

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity., info:eu-repo/semantics/published

Hereditary motor neuropathies are clinically and genetically diverse disorders characterized by length-dependent axonal degeneration of lower motor neurons. Although currently as many as 26 causal genes are known, there is considerable missing heritability compared to other inherited neuropathies such as Charcot-Marie-Tooth disease. Intriguingly, this genetic landscape spans a discrete number of key biological processes within the peripheral nerve. Also, in terms of underlying pathophysiology, hereditary motor neuropathies show striking overlap with several other neuromuscular and neurological disorders. In this review, we provide a current overview of the genetic spectrum of hereditary motor neuropathies highlighting recent reports of novel genes and mutations or recent discoveries in the underlying disease mechanisms. In addition, we link hereditary motor neuropathies with various related disorders by addressing the main affected pathways of disease divided into five major processes: axonal transport, tRNA aminoacylation, RNA metabolism and DNA integrity, ion channels and transporters and endoplasmic reticulum.

When the primitive translation system first emerged in the hypothetical RNA world, ribozymes could have been responsible for aminoacylation. Given that naturally occurring T-box riboswitches selectively sense the aminoacylation status of cognate tRNAs, we introduced a domain of random sequence into a T-box–tRNA conjugate and isolated ribozymes that were self-aminoacylating on the 3′-terminal hydroxyl group. One of them, named Tx2.1, recognizes the anticodon and D-loop of tRNA via interaction with its stem I domain, similarly to the parental T-box, and selectively charges N-biotinyl-l-phenylalanine (Bio-lPhe) onto the 3′ end of the cognate tRNA in trans. We also demonstrated the ribosomal synthesis of a Bio-lPhe-initiated peptide in a Tx2.1-coupled in vitro translation system, in which Tx2.1 catalyzed specific tRNA aminoacylation in situ. This suggests that such ribozymes could have coevolved with a primitive translation system in the RNA world. The authors develop a ribozyme, Tx2.1, that is capable of aminoacylating tRNA with specificity for the anticodon from directed evolution of a T-box riboswitch. Tx2.1 could be used to charge non-natural amino acids in an in vitro translation system.

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases (LysRSs). Previously, two mutations (c.1129G>A and c.517T>C) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNALys, with the c.517T>C mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.

β-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS–purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chains intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting proteins safe passage to their native states; however, the complexity of this medium has generally precluded biophysical techniques from interrogating protein folding under cellular-like conditions for single proteins, let alone entire proteomes. Here, we develop a limited-proteolysis mass spectrometry approach paired within an isotope-labeling strategy to globally monitor the structures of refolding E. coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data, and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than indirect measures like binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones both unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. The data support a model in which chaperone-nonrefolders have evolved to fold efficiently once and only once, co-translationally, and remain kinetically trapped in their native conformations. HIGHLIGHTS- New proteomic methods are developed to probe protein refolding globally and sensitively in a complex background - The results revise the consensus model of which E. coli proteins require the GroEL chaperonin for efficient refolding - DnaK (Hsp70) and GroEL refold largely the same clientele, suggesting that these distinct chaperones share a common unifying mechanism - A small cohort of proteins cannot be fully restored by chaperones. These chaperone- nonrefolders tend to be involved in tRNA aminoacylation and glycolysis, and may have kinetically-trapped native states

Lung cancer, one of the most malignant tumors, has extremely high morbidity and mortality, posing a serious threat to global health. It is an urgent need to fully understand the pathogenesis of lung cancer and provide new ideas for its treatment. Interestingly, accumulating evidence has identified that transfer RNAs (tRNAs) and tRNA metabolism–associated enzymes not only participate in the protein translation but also play an important role in the occurrence and development of lung cancer. In this review, we summarize the different aspects of tRNA metabolism in lung cancer, such as tRNA transcription and mutation, tRNA molecules and derivatives, tRNA-modifying enzymes, and aminoacyl-tRNA synthetases (ARSs), aiming at a better understanding of the pathogenesis of lung cancer and providing new therapeutic strategies for it.

Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity. IMPORTANCE Most bacteria use a two-step indirect pathway to aminoacylate tRNAGln and tRNAAsn, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.

Uvod: Amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD) sta hitro napredujoči, neozdravljivi in smrtonosni nevrodegenerativni bolezni. Najpogostejši genetski vzrok ALS in FTD je mutacija v genu C9orf72 (angl. chromosome 9 open reading frame 72). Mutacija se kaže v obliki razširjenih ponovitev zapisa G4C2, katerih število pri obolenju doseže več 100 lahko tudi več 1000 ponovitev. Pri ALS in FTD naj bi mutacija povzročala: haploinsuficienco proteina C9orf72, z RNA posredovano toksičnost smernih in protismernih prepisov RNA razširjenih ponovitev ter toksičnost proteinov z dipeptidnimi ponovitvami (DPR), ki se prevedejo iz smernih in protismernih zapisov RNA. Z RNA posredovana toksičnost je posledica sekvestracije proteinov na skupke smernih in protismernih zapisov RNA, kar onemogoči njihove normalne funkcije v celici. Obstaja veliko raziskav o vezavi različnih proteinov na smerne zapise RNA. Vezava proteinov na protismerne zapise je raziskana v veliko manjši meri. Veliko raziskav je usmerjenih tudi v preučevanje DPR-jev in z njimi povezanih okvar celičnih procesov. Vse več pozornosti se namenja tudi povezavi med vsemi tremi mehanizmi, ki jih povzroča mutacija v genu C9orf72 ter na njihovo skupno vlogo v patologiji bolezni. Namen dela in hipoteza: Tako smerni kot protismerni zapisi RNA razširjenih ponovitev G4C2 v genu C9orf72 vežejo nase različne proteine, kar prispeva k patologiji ALS in FTD. Ker so v večji meri raziskani le smerni zapisi RNA smo se v naši raziskavi osredotočili na manj raziskane protismerne zapise RNA ponovitev v genu C9orf72. Hipoteza, na kateri sloni raziskava, se glasi: Protismerni zapis RNA mutacije v genu C9orf72 veže za celico pomembne proteine, kar je lahko povezano s patologijo ALS in FTD. Namen dela je bil identificirati proteine, ki vežejo protismerni zapis RNA razširjenih ponovitev mutacije v genu C9orf72 ter opredeliti potencialno vlogo teh interakcij pri patologiji ALS in FTD. Poleg tega smo v nalogi ovrednotili vpliv proteina SFPQ (angl. splicing factor proline and glutamine rich), za katerega je znano, da veže smerne zapise RNA ponovitev v genu C9orf72, na število jedrnih skupkov tako smernih kot protismernih zapisov RNA ponovitev v genu C9orf72 ter na raven izražanja DPR-jev. Metode dela: Za identifikacijo proteinov, ki vežejo dolge ponovitve protismerne RNA iz razširjenih ponovitev G4C2 v genu C9orf72, smo vzpostavili test RNA “pull down”. Interakcijo protismerne RNA z identificiranimi proteini smo preverili na celicah s prisotno mutacijo v genu C9orf72 s pomočjo RNA fluorescenčne in situ hibridizacije (RNA-FISH) v kombinaciji z imunocitokemijo (ICC) in analizo kolokalizacije fluorescenčnega signala skupkov RNA in izbranih proteinov. Kot prvi smo optimizirali metodo RNA-protein PLA (angl. proximity ligation assay) za analizo interakcij protismernih ponovitev RNA in proteinov v citoplazmi tudi izven znanih jedrnih skupkov RNA. Optimizirali smo protokol za preverjanje stopnje aminoacilacije tRNA v kombinaciji s kvantitativno verižno reakcijo s polimerazo (qPCR) in naše rezultate uspešno analizirali z metodo ddCt. Vpliv ravni izražanja proteina SFPQ na število skupkov RNA in na izražanje DPR-jev v celicah smo ovrednotili v vzpostavljenih celičnih modelih s prekomerno izraženim ali utišanim proteinom SFPQ. V teh celičnih modelih smo izražali smerne oziroma protismerne zapise razširjenih ponovitev gena C9orf72. Za prekomerno izražanje proteina SFPQ smo uporabili transfekcijo, za njegovo utišanje pa transdukcijo celic z lentivirusnimi delci. Raven izražanja A podenote fenilalanin-tRNA sintetaze (FARSA), SFPQ in DPR-jev smo preverili s prenosom western in točkovnim nanosom vzorcev z analizo intenzitete končnega protitelo-antigen signala. Detekcijo posameznih skupkov RNA in proteinov ter signala RNA-protein z metodo PLA smo izvedli s konfokalno fluorescenčno mikroskopijo. Kvantifikacija je bila izvedena s programom ImageJ. Za statistično analizo smo uporabili Studentov t-test. Rezultati in razprava: Pokazali smo, da C4G2 ponovitve RNA vežejo proteine vpletene v različne celične procese. Identificirani proteini sodelujejo pri sestavi in stabilnosti citoskeleta, transportu RNA in proteinov po aksonih in dendritih (CYFIP1/2, TAOK1), pri procesiranju in transportu molekul RNA (HNRNPL), pri sintezi proteinov (FARSA/B), pri funkciji mielinizirajočih celic (CNP), pri biogenezi ribosomov in pri preverjanju kvalitete proteinov (NPM1). Identificirani proteini so bili že povezani z različnimi nevrodegenerativnimi boleznimi, med njimi tudi z ALS. V nadaljevanju raziskave smo se osredotočili na citoplazemski interaktor fenilalanin-tRNA sintetazo (FARS), ki je sestavljena iz podenote ? (FARSA), katere naloga je vezava ustrezne aminokisline na tRNA in podenote ß (FARSB), katere naloga je odstranitev nepravilno pripete aminokisline iz tRNA. Z namenom preučevanja citoplazemskih interakcij protismerne RNA C4G2 s proteini v celičnih modelih, smo optimizirali protokol RNA-protein PLA detekcije. Interakcije RNA C4G2 s FARSA smo uspeli potrditi v treh različnih celičnih linijah z mutacijo v genu C9orf72 (fibroblastih, limfoblastih in iPSC-ih). Okvare v delovanju FARSA povzročene z vezavo na C4G2 RNA bi lahko vodile v znižano aminoacilacijo Phe-tRNA. Zato smo optimizirali protokol za določanje stopnje aminoacilacije tRNA, s katerim smo v limfoblastih s prisotno mutacijo v genu C9orf72 odkrili bistveno znižanje ravni aminoacilacije Phe-tRNA, v primerjavi s kontrolnimi limfoblasti. Napake v delovanju aminoacil-tRNA sintetaz (ARS) lahko vodijo v napačno zvijanje in kopičenje proteinov, kar je značilno za nevrodegenerativne bolezni. Poleg tega imajo ARS-i še veliko nestandardnih funkcij, ki bi bile lahko prav tako prizadete ob interakciji teh proteinov s ponovitvami C4G2 RNA v celicah. V drugem delu naloge smo proučili vpliv proteina SFPQ, povezanega z nevrodegeneracijo, na tvorbo skupkov smerne in protismerne RNA razširjenih ponovitev gena C9orf72 in na sintezo DPR-jev. Znižana raven izražanja proteina SFPQ zniža število skupkov smerne in v manjši meri tudi protismerne RNA v celici, ter zniža raven izražanja DPR-jev. Prekomerno izražanje proteina SFPQ pa poveča število skupkov smerne in protismerne RNA ter sintezo DPR-jev. SFPQ je znan interaktor smerne RNA razširjenih ponovitev gena C9orf72, mi pa smo pokazali, da ne veže protismerne RNA razširjenih ponovitev gena C9orf72. S tem lahko razložimo manjši vpliv ravni izražanja SFPQ na skupke protismerne RNA. Pokazano je bilo, da protein SFPQ uravnava prepisovanje genov, ki imajo kompleksno sekundarno strukturo na ravni DNA. Predvidevamo, da posledično lahko vpliva tudi na prepisovanje razširjenih ponovitev gena C9orf72, ki prav tako tvorijo sekundarne strukture na ravni DNA. To pojasni vpliv ravni izražanja proteina SFPQ na število tako smernih kot protismernih skupkov RNA ponovitev C9orf72 ter tudi na sintezo DPR-jev, ki smo jo opazili. Spremenjena sinteza DPR-jev pa bi bila lahko uravnavana tudi na ravni translacije, saj je SFPQ vpleten tudi v mehanizme translacije. Zaključki in znanstveno-raziskovalni pomen študije: Določili smo nove proteine, ki vežejo manj raziskane protismerne zapise RNA razširjenih ponovitev gena C9orf72, ki so vpleteni v različne celične procese. Okvare funkcije teh proteinov zaradi njihove vezave na protismerno RNA bi lahko imele značilne posledice za delovanje nevronov. Prvi smo odkrili interakcijo proteina FARS s protismernimi RNA razširjenih ponovitev gena C9orf72 in okvaro aminoacilacije tRNA v celicah z mutacijo v genu C9orf72 in tako pomembno prispevali k razumevanju okvare mehanizmov in prizadetosti celičnih procesov pri ALS in FTD s prisotno mutacijo v genu C9orf72. Odkriti mehanizem odpira nove možnosti za terapevtske pristope pri ALS in FTD obolenjih. Za ovrednotenje citoplazemske interakcije protismerne RNA, smo prvi v ta namen optimizirali in uporabili metodo RNA-protein PLA. Do sedaj so metode omogočale le analize interakcij razširjenih ponovitev RNA znotraj jedra. Z našo optimizirano RNA-protein PLA metodo pa smo omogočili tudi analize interakcij v citoplazmi. S tem smo pomembno prispevali k metodološkem pristopu analize interakcij razširjenih ponovitev RNA mutacije v genu C9orf72 z različnimi celičnimi proteini. Prav tako smo pokazali, da raven izražanja proteina SFPQ vpliva na tvorbo skupkov tako smerne kot protismerne RNA in na sintezo DPR-jev. S tem smo pripevali k razumevanju vloge proteina SFPQ pri bolezni ALS in FTD in njegovega vpliva na toksične mehanizme, ki jih mutacija v genu C9orf72 proži. Uravnavanje ravni SFPQ-ja in z njim povezanih mehanizmov je potencialen terapevtski pristop za zdravljenje ALS in FTD s prisotno mutacijo v genu C9orf72. Introduction: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fast progressive and fatal neurodegenerative diseases. The most common genetic cause of both diseases is mutation in the chromosome 9 open reading frame 72 gene (C9orf72). The mutation is expanded repeat of G4C2 sequence. In disease the number of repeats reaches more hundreds and more thousand units. There are three proposed mechanisms of action for the mutation: haploinsufficiency of C9orf72 protein, RNA toxicity exerted by sense and antisense RNA repeats, and toxicity of dipeptide repeat proteins (DPRs) translated from the sense and antisense RNA repeats. RNA toxicity is the consequence of sequestration of RNA-binding proteins by sense and antisense RNA repeats, which impairs their normal cellular function. Many studies aimed to identify proteins binding to sense RNA repeats however, there is a distinct lack of studies of antisense RNA repeats. Another major subject of the studies related to the C9orf72 mutation are DPRs and their influence on cell mechanisms. The prevailing view is that all C9orf72 mutation mechanisms work together in disease pathology. Aims and Hypothesis: Both sense and antisense RNA transcripts from C9orf72 mutation bind various proteins, which contributes to the pathology of ALS and FTD. This research is focused on the less studied antisense RNA. The study is based on the following hypothesis: Antisense RNA transcripts of C9orf72 gene mutation bind important cellular proteins and these interactions may be associated with pathology of ALS and FTD. Our aim was to identify proteins binding to antisense RNA repeats and define their potential role in disease pathology. Moreover, we also wanted to define the impact of known sense RNA interactor splicing factor proline and glutamine rich (SFPQ) on the number of sense and antisense RNA foci and DPR expression levels, as we propose it impacts stability and formation of RNA foci and, consequently, DPR production. Methods: In order to identify the proteins binding to long antisense repeats, we set up RNA pull-down experiment. We studied the antisense RNA-protein colocalizations in C9orf72 mutation-positive patient-derived cells using RNA fluorescent in situ hybridization (RNA-FISH) in combination with immunocytochemistry (ICC) with overlap analysis of RNA foci and protein fluorescence signals. We were first to establish RNA-protein proximity ligation assay (PLA) for detection of interactions outside of nuclear RNA foci. To analyze the catalytic function of Phe-tRNA synthetase (FARS) in C9orf72 mutation, we optimized the tRNA aminoacylation assay in combination with quantitative polymerase chain reaction (qPCR). We analyzed the results with ddCt method. Furthermore, we studied the impact of SFPQ on sense and antisense RNA foci and DPRs by either SFPQ overexpression or knockdown in the cells expressing sense or antisense RNA repeats. Transfection was used for the protein overexpression and lentiviral transduction for the SFPQ knockdown in cells. The expression levels of FARSA, SFPQ, and DPRs were analyzed by western blot and dot blot. Densitometric analyses of the protein signals were performed. The fluorescently labeled RNA foci and proteins, and RNA-protein PLA signals in cells were analyzed by confocal microscopy. We used ImageJ for RNA foci and RNA-protein PLA signal counts. Statistical significance was calculated with the unpaired, two-tailed Student’s t-test. Results and discussion: In this research, we identified various new proteins interacting with C4G2 RNA, which are implicated in multiple cellular mechanisms including composition and stability of cytoskeleton, axonal and dendritic transport (CYFIP1/2, TAOK1), RNA processing and transport (HNRNPL), protein synthesis (FARSA/B), myelinating cell functions (CNP), ribosome biogenesis and protein quality control (NPM1). The identified proteins have been previously associated with various neurodegenerative diseases, including ALS and FTD. We focused on the cytoplasmic interactor –FARS. FARS is composed of two subunits, FARSA is responsible for the attachment of cognate amino acid to its tRNA and FARSB has an editing function for elimination of amino acids from misacylated tRNAs. In order to study cytoplasmic interactions of antisense RNA with identified proteins, we optimized RNA-protein PLA method. We confirmed interactions of FARSA with antisense RNA in three C9orf72 mutation-positive patient-derived cell lines (fibroblasts, lymphoblasts and iPSCs) with RNA-protein PLA. Furthermore, in order to study the catalytic function of FARSA in C9orf72 mutation, we optimized the tRNA aminoacylation assay. We discovered there is a significant decrease of charged Phe-tRNA in C9orf72 mutation-positive patient-derived lymphoblasts compared to controls. Disruptions of aminoacyl-tRNA synthetases (ARS) have been previously shown to cause protein misfolding and accumulation, and are implicated in various disorders of neurological system. Moreover, ARSs have multiple uncanonical functions, which could also be affected and play role in multiple diseases. We also investigated the role of SFPQ in the formation of both sense and antisense RNA foci and the production of DPRs. SFPQ has been previously implicated in ALS and FTD and is a known interactor of sense RNA. Reduction in SFPQ expression levels led to reduction in the number of sense and antisense RNA foci and in the expression levels of DPRs. SFPQ overexpression increased the numbers of sense and antisense RNA foci and the DPRs production. Overall, the impact on antisense RNA foci was lower compared to sense RNA foci. We showed that, in contrast to sense RNA, SFPQ does not bind to antisense RNA, which could explain this effect. Nevertheless, the impact on numbers of antisense RNA foci was still significant, which could be the consequence of SFPQ involvement in transcription regulation of the C9orf72 expanded repeats. It was previously shown that SFPQ enables transcription of genes with complex secondary structures, which makes it a good candidate for transcription of the C9orf72 repeat expansion, which form various secondary structures on DNA and RNA level. This could also explain the consequential impact on the DPRs` levels. However, the impact on DPRs could also be on the account of SPFQ involvement in translational regulation, which was also previously shown. Conclusions and scientific significance of the study: We identified multiple new interactors of less studied antisense RNA. Identified proteins are involved in multiple cell processes, and impairments in their function on account of their sequestration could be detrimental for neurons. We are first to identify the involvement of FARS and impairment of tRNA aminoacylation in C9orf72 mutation. Therefore, we significantly contributed to the understanding of the mechanisms impaired in the C9orf72 ALS and FTD. Furthermore, the involvement of tRNA aminoacylation in disease mechanisms opens new targets for therapeutic approaches. We are first to optimize the RNA-protein PLA method for detection of cytoplasmic interactions of antisense RNA. This is an important methodological contribution to the field of studying protein interactors of repeat expansion RNAs in cells, as these were so far limited to colocalizations of proteins with nuclear RNA foci. We also evaluated the impact of SFPQ expression levels on both sense and antisense RNA foci formation and on DPR synthesis. Therefore, we expanded the knowledge on the role of formerly known ALS and FTD protein SFPQ in C9orf72 mutation toxic mechanisms. The modulation of SFPQ expression level or its associated pathways represents a potential therapeutic approach in the C9orf72 ALS and FTD.

Nucleotides of transfer RNAs (tRNAs) are highly modified, particularly at the anticodon. Bacterial tRNAs that read A-ending codons are especially notable. The U34 nucleotide canonically present in these tRNAs is modified by a wide range of complex chemical constituents. An additional two A-ending codons are not read by U34-containing tRNAs but are accommodated by either inosine or lysidine at the wobble position (I34 or L34). The structural basis for many N34 modifications in both tRNA aminoacylation and ribosome decoding has been elucidated, and evolutionary conservation of modifying enzymes is also becoming clearer. Here we present a brief review of the structure, function, and conservation of wobble modifications in tRNAs that translate A-ending codons. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1158-1166, 2019.

Aminoacyl-tRNA synthetases (AARSs) charge their cognate tRNAs with corresponding amino acids, playing key roles in ribosomal protein synthesis. A series of proteomic studies have demonstrated that AARSs have levels of lysine acetylation much higher than those of other proteins in Escherichia coli. To study AARS acetylation, 25 site-specifically acetylated variants of four AARSs were generated by the genetic code expansion strategy. Kinetic analyses were performed to biochemically characterize the impact of site-specific acetylation on AARS functions, including amino acid activation, tRNA aminoacylation, and editing activities. The results showed that impacts of acetylation were different between class I and class II AARSs and also varied among the same class of AARSs. The results also showed that acetylation of threonyl-tRNA synthetase (ThrRS) could affect its editing function. Both in vivo and in vitro studies were further performed to explore the acetylation and deacetylation processes of ThrRS. Although nonenzymatic acetylation and CobB-dependent deacetylation were concluded, the results also indicated the existence of additional modifying enzymes or mechanisms for ThrRS acetylation and deacetylation.

N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E. coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the A site. Enzyme and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the starvation-induced stringent response, consistent with the notion that loss of tRNA aminoacylation activates the response. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.

Transfer RNAs (tRNAs) mainly function as adapter molecules that decode messenger RNAs (mRNAs) during protein translation by delivering amino acids to the ribosome. Traditionally, tRNAs are considered as housekeepers without additional functions. Nevertheless, it has become apparent from biological research that tRNAs are involved in various physiological and pathological processes. Aging is a form of gradual decline in physiological function that ultimately leads to increased vulnerability to multiple chronic diseases and death. Interestingly, tRNA metabolism is closely associated with aging and lifespan. In this review, we summarize the emerging roles of tRNA-associated metabolism, such as tRNA transcription, tRNA molecules, tRNA modifications, tRNA aminoacylation, and tRNA derivatives, in aging and lifespan, aiming to provide new ideas for developing therapeutics and ultimately extending lifespan in humans.

Aminoacyl-RNA synthetases (aaRSs) are among the key enzymes of protein biosynthesis. They are responsible for conducting the first step in the protein biosynthesis, namely attaching amino acids to the corresponding tRNA molecules both in cytoplasm and mitochondria. More and more research demonstrates that mutations in the genes encoding aaRSs lead to the development of various neurodegenerative diseases, such as incurable Charcot–Marie–Tooth disease (CMT) and distal spinal muscular atrophy. Some mutations result in the loss of tRNA aminoacylation activity, while other mutants retain their classical enzyme activity. In the latter case, disease manifestations are associated with additional neuron-specific functions of aaRSs. At present, seven aaRSs (GlyRS, TyrRS, AlaRS, HisRS, TrpRS, MetRS, and LysRS) are known to be involved in the CMT etiology with glycyl-tRNA synthetase (GlyRS) being the most studied of them.

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 A. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys.

Mammalian mitochondrial tRNA (mt-tRNA) plays a central role in the synthesis of the 13 subunits of the oxidative phosphorylation complex system (OXPHOS). However, many aspects of the context-dependent expression of mt-tRNAs in mammals remain unknown. To investigate the tissue-specific effects of mt-tRNAs, we performed a comprehensive analysis of mitochondrial tRNA expression across five mice tissues (brain, heart, liver, skeletal muscle, and kidney) using Northern blot analysis. Striking differences in the tissue-specific expression of 22 mt-tRNAs were observed, in some cases differing by as much as tenfold from lowest to highest expression levels among these five tissues. Overall, the heart exhibited the highest levels of mt-tRNAs, while the liver displayed markedly lower levels. Variations in the levels of mt-tRNAs showed significant correlations with total mitochondrial DNA (mtDNA) contents in these tissues. However, there were no significant differences observed in the 2-thiouridylation levels of tRNALys, tRNAGlu, and tRNAGln among these tissues. A wide range of aminoacylation levels for 15 mt-tRNAs occurred among these five tissues, with skeletal muscle and kidneys most notably displaying the highest and lowest tRNA aminoacylation levels, respectively. Among these tissues, there was a negative correlation between variations in mt-tRNA aminoacylation levels and corresponding variations in mitochondrial tRNA synthetases (mt-aaRS) expression levels. Furthermore, the variable levels of OXPHOS subunits, as encoded by mtDNA or nuclear genes, may reflect differences in relative functional emphasis for mitochondria in each tissue. Our findings provide new insight into the mechanism of mt-tRNA tissue-specific effects on oxidative phosphorylation.

The global rise in type 2 diabetes results from a combination of genetic predisposition with environmental assaults that negatively affect insulin action in peripheral tissues and impair pancreatic β-cell function and survival. Nongenetic heritability of metabolic traits may be an important contributor to the diabetes epidemic. Transfer RNAs (tRNAs) are noncoding RNA molecules that play a crucial role in protein synthesis. tRNAs also have noncanonical functions through which they control a variety of biological processes. Genetic and environmental effects on tRNAs have emerged as novel contributors to the pathogenesis of diabetes. Indeed, altered tRNA aminoacylation, modification, and fragmentation are associated with β-cell failure, obesity, and insulin resistance. Moreover, diet-induced tRNA fragments have been linked with intergenerational inheritance of metabolic traits. Here, we provide a comprehensive review of how perturbations in tRNA biology play a role in the pathogenesis of monogenic and type 2 diabetes., info:eu-repo/semantics/published

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., SCOPUS: ar.j, info:eu-repo/semantics/published

Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3&prime, 32P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the &alpha, amine or the &alpha, imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3&prime, 32P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.

Most bacteria, including mycobacteria, utilize a two-step indirect tRNA aminoacylation pathway to generate correctly aminoacylated glutaminyl and asparaginyl tRNAs. This involves an initial step in which a non-discriminatory aminoacyl tRNA synthetase misacylates the tRNA, followed by a second step in which the essential amidotransferase, GatCAB, amidates the misacylated tRNA to its correct, cognate form. It had been previously demonstrated that mutations in gatA can mediate increased error rates specifically of glutamine to glutamate or asparagine to aspartate in protein synthesis. However, the role of mutations in gatB or gatC in mediating mistranslation are unknown. Here, we applied a forward genetic screen to enrich for mistranslating mutants of Mycobacterium smegmatis. The majority (57/67) of mutants had mutations in one of the gatCAB genes. Intriguingly, the most common mutation identified was an insertion in the 3' of gatC, abolishing its stop codon, and resulting in a fused GatC-GatA polypeptide. Modeling the effect of the fusion on GatCAB structure suggested a disruption of the interaction of GatB with the CCA-tail of the misacylated tRNA, suggesting a potential mechanism by which this mutation may mediate increased translational errors. Furthermore, we confirm that the majority of mutations in gatCAB that result in increased mistranslation also cause increased tolerance to rifampicin, although there was not a perfect correlation between mistranslation rates and degree of tolerance. Overall, our study identifies that mutations in all three gatCAB genes can mediate adaptive mistranslation and that mycobacteria are extremely tolerant to perturbation in the indirect tRNA aminoacylation pathway.

Paradoxically, oncogenes that drive cell cycle progression may also trigger pathways leading to senescence, thereby inhibiting the growth of tumorigenic cells. Along these lines, Y1 cells, which carry an amplification of Ras, become senescent after treatment with the mitogen FGF-2. To understand how FGF-2 promotes senescence, we profiled the epigenome, transcriptome, proteome, and phospho-proteome of Y1 cells stimulated with FGF-2. FGF-2 caused delayed acetylation of histone H4 and higher levels of H3K27me3. Sequencing analysis revealed decreased expression of cell cycle-related genes with concomitant loss of H3K27ac. In contrast, FGF-2 promoted the expression of p21, various cytokines, and MAPK-related genes. Nuclear envelope proteins, particularly lamin B1, displayed increased phosphorylation in response to FGF-2. Proteome analysis suggested alterations in cellular metabolism, as evident by modulated expression of enzymes involved in purine biosynthesis, tRNA aminoacylation, and the TCA cycle. Altogether, the response of Y1 cells to FGF-2 is consistent with oncogene-induced senescence. We propose that Y1 cells enter senescence due to deficient cyclin expression and high levels of p21, which may stem from DNA damage or TGFb signaling.

Author(s): Won, Eric; Morodomi, Yosuke; Kanaji, Sachiko; Shapiro, Ryan; Vo, My-Nuong; Orje, Jennifer N; Thornburg, Courtney D; Yang, Xiang-Lei; Ruggeri, Zaverio M; Schimmel, Paul; Kanaji, Taisuke | Abstract: BackgroundTyrosyl-tRNA synthetase (YRS) belongs to the family of enzymes that catalyzes the tRNA aminoacylation reaction for protein synthesis, and it has been recently shown to exert noncanonical functions. Although database results indicate extremely low levels of YRS mRNA in platelets, YRS protein is abundantly present. The source of YRS in platelets, as well as the physiological role of platelet-stored YRS, remains largely unknown.ObjectivesTo clarify how YRS accumulates in platelets and determine the potential role of platelet-stored YRS.MethodsRecombinant YRS proteins with epitope tags were prepared and tested in vitro for proteolytic cleavage in human plasma. Fluorescent-labeled YRS was examined for uptake by platelets, as demonstrated by western blotting and confocal microscopy analysis. Using RAW-Dual reporter cells, Toll-like receptor and type I interferon activation pathways were analyzed after treatment with YRS.ResultsFull-length YRS was cleaved by both elastase and matrix metalloproteinases in the plasma. The cleaved, N-terminal YRS fragment corresponds to the endogenous YRS detected in platelet lysate by western blotting. Both full-length and cleaved forms of YRS were taken up by platelets in vitro and stored in the α-granules. The N-terminal YRS fragment generated by proteolytic cleavage had monocyte activation comparable to that of the constitutive-active mutant YRS (YRSY341A) previously reported.ConclusionPlatelets take up both full-length YRS and the active form of cleaved YRS fragment from the plasma. The cleaved, N-terminal YRS fragment stored in α-granules may have potential to activate monocytes.

Leber’s Hereditary Optic Neuropathy (LHON) was a common maternally inherited disease causing severe and permanent visual loss which mostly affects males. Three primary mitochondrial DNA (mtDNA) mutations, ND1 3460G>A, ND4 11778G>A and ND6 14484T>C, which affect genes encoding respiratory chain complex I subunit, are responsible for >90% of LHON cases worldwide. Families with maternally transmitted LHON show incomplete penetrance with a male preponderance for visual loss, suggesting the involvement of secondary mtDNA variants and other modifying factors. In particular, variants in mitochondrial tRNA (mt-tRNA) are important risk factors for LHON. These variants decreased the tRNA stability, prevent tRNA aminoacylation, influence the post-transcriptionalmodification and affect tRNA maturation. Failure of mt-tRNA metabolism subsequently impairs protein synthesis and expression, folding, and function of oxidative phosphorylation (OXPHOS) enzymes, which aggravates mitochondrial dysfunction that is involved in the progression and pathogenesis of LHON. This review summarizes the recent advances in our understanding of mt-tRNA biology and function, as well as the reported LHON-related mt-tRNA second variants; it also discusses the molecular mechanism behind the involvement of these variants in LHON.

RNase Z is a widely distributed and usually essential endoribonuclease involved in the 3′-end maturation of transfer RNAs (tRNAs). A CCA triplet that is needed for tRNA aminoacylation in protein translation is added by a nucleotidyl-transferase after the 3′-end processing by RNase Z. However, a considerable proportion of the archaeal pre-tRNAs genetically encode a CCA motif, while the enzymatic characteristics of the archaeal RNase (aRNase) Zs in processing CCA-containing pre-tRNAs remain unclear. This study intensively characterized two methanomicrobial aRNase Zs, the Methanolobus psychrophilus mpy-RNase Z and the Methanococcus maripaludis mmp-RNase Z, particularly focusing on the properties of processing the CCA-containing pre-tRNAs, and in parallel comparison with a bacterial bsu-RNase Z from Bacillus subtilis. Kinetic analysis found that Co2+ supplementation enhanced the cleavage efficiency of mpy-RNase Z, mmp-RNase Z, and bsu-RNase Z for 1400-, 2990-, and 34-fold, respectively, and Co2+ is even more indispensable to the aRNase Zs than to bsu-RNase Z. Mg2+ also elevated the initial cleavage velocity (V0) of bsu-RNase Z for 60.5-fold. The two aRNase Zs exhibited indiscriminate efficiencies in processing CCA-containing vs. CCA-less pre-tRNAs. However, V0 of bsu-RNase Z was markedly reduced for 1520-fold by the CCA motif present in pre-tRNAs under Mg2+ supplementation, but only 5.8-fold reduced under Co2+ supplementation, suggesting Co2+ could ameliorate the CCA motif inhibition on bsu-RNase Z. By 3′-RACE, we determined that the aRNase Zs cleaved just downstream the discriminator nucleotide and the CCA triplet in CCA-less and CCA-containing pre-tRNAs, thus exposing the 3′-end for linking CCA and the genetically encoded CCA triplet, respectively. The aRNase Zs, but not bsu-RNase Z, were also able to process the intron-embedded archaeal pre-tRNAs, and even process pre-tRNAs that lack the D, T, or anticodon arm, but strictly required the acceptor stem. In summary, the two methanomicrobial aRNase Zs use cobalt as a metal ligand and process a broad spectrum of pre-tRNAs, and the characteristics would extend our understandings on aRNase Zs.

Aminoacyl-tRNA synthetases (AARSs) play key roles in maintaining high fidelity of protein synthesis. They charge cognate tRNAs with corresponding amino acids and hydrolyze mischarged tRNAs by editing mechanisms. Impairment of AARS editing activities can reduce the accuracy of tRNA aminoacylation to produce mischarged tRNAs, which cause mistranslation and cell damages. To evaluate the mistranslation rate of threonine codons in living cells, in this study, we designed a quantitative reporter derived from the green fluorescent protein (GFP). The original GFP has multiple threonine codons which could affect the accuracy of measurement, so we generated a GFP variant containing only one threonine residue to specifically quantify mistranslation at the threonine codon. To validate, we applied this single-threonine GFP reporter to evaluate mistranslation at the threonine codon with mutations or modifications of threonine-tRNA synthetase and compared it with other methods of mistranslation evaluation, which showed that this reporter is reliable and facile to use.

Visual Abstract, Alzheimer’s disease (AD) starts decades before clinical symptoms appear. Low-glucose utilization in regions of the cerebral cortex marks early AD. To identify these regions, we conducted a voxel-wise meta-analysis of previous studies conducted with positron emission tomography that compared AD patients with healthy controls. The resulting map marks hypometabolism in the posterior cingulate, middle frontal, angular gyrus, and middle and inferior temporal regions. Using the Allen Human Brain Atlas, we identified genes that show spatial correlation across the cerebral cortex between their expression and this hypometabolism. Of the six brains in the Atlas, one demonstrated a strong spatial correlation between gene expression and hypometabolism. Previous neuropathological assessment of this brain from a 39-year-old male noted a neurofibrillary tangle in the entorhinal cortex. Using the transcriptomic data, we estimate lower proportions of neurons and more microglia in the hypometabolic regions when comparing this donor’s brain with the other five donors. Within this single brain, signal recognition particle (SRP)-dependent cotranslational protein targeting genes, which encode primarily cytosolic ribosome proteins, are highly expressed in the hypometabolic regions. Analyses of human and mouse data show that expression of these genes increases progressively across AD-associated states of microglial activation. In addition, genes involved in cell killing, chronic inflammation, ubiquitination, tRNA aminoacylation, and vacuole sorting are associated with the hypometabolism map. These genes suggest disruption of the protein life cycle and neuroimmune activation. Taken together, our molecular characterization reveals a link to AD-associated hypometabolism that may be relevant to preclinical stages of AD.

Background Excessive or prolonged usage of dexamethasone can cause serious side effects, but few studies reveal the related mechanism. Dexamethasone work differently in blood tumors and solid tumors, and the cause is still obscure. The aims of this study was to identify potential biomarkers associated with the side effects of dexamethasone in different tumors. Methods Gene Expression Omnibus database (GEO) datasets of blood tumors and solid tumors were retrieval to selected microarray data. The differentially expressed genes (DEGs) were identified. Gene ontology (GO) and pathway enrichment analyses, and protein–protein interaction (PPI) network analysis were performed. Results One hundred and eighty dexamethasone‐specific DEGs (92 up and 88 downregulated) were obtained in lymphoma cell samples (named as DEGs‐lymph), including APOD, TP53INP1, CLIC3, SERPINA9, and C3orf52. One hundred and four specific DEGs (100 up and 4 downregulated) were identified in prostate cancer cell samples (named as DEGs‐prostate), including COL6A2, OSBPL5, OLAH, OGFRL1, and SLC39A14. The significantly enriched GO terms of DEGs‐lymph contained cellular amino acid metabolic process and cell cycle. The most significantly enriched pathway of DEGs‐lymph was cytosolic tRNA aminoacylation. The DEGs‐prostate was enriched in 39 GO terms and two pathways, and the pathways were PPARA activates gene expression Homo sapiens, and insulin resistance. The PPI network of DEGs‐lymph gathered into two major clusters, WARS1 and CDC25A were representatives for them, respectively. One cluster was mainly involved in cytosolic tRNA aminoacylation, aminoacyl‐tRNA biosynthesis and the function of amino acid metabolism; another was associated with cell cycle and cell apoptosis. As for the PPI network of DEGs‐prostate, HELZ2 was the top nodes involved in the most protein–protein pairs, which was related to the pathway of “PPARA activates gene expression Homo sapiens.” Conclusions WARS1 and CDC25A might be potential biomarkers for side effects of dexamethasone in lymphoma, and HELZ2 in prostate cancer., The aims of this study was to identify potential biomarkers associated with the side effects of dexamethasone in different tumors. WARS and CDC25A might be potential biomarkers for side effects of dexamethasone in lymphoma, and HELZ2 in prostate cancer.

Currently, the cell and tissue storage using the cryoprotective agents are quite common, in particular in reproductive technologies. Meanwhile the issue of safety when applying the CPAs remains open, since even in residual amounts after washing, they can affect the functioning of the most critical metabolic processes of a cell, in particular transcription and translation, which can be of great importance for further life and development of organs, tissues, cells. The goal was to study the effect of penetrating cryoprotective agents glycerol, ME2SO, ethylene glycol, and non-penetrating PEG-400 on protein synthesizing activity in cell-free systems of Krebs-2 ascites carcinoma and wheat germ. In this study, we compared the effects of ME2SO, PEG-400, glycerol, and ethylene glycol on protein biosynthesis in cell-free systems according to the incorporation of 14C-amino acids in total proteins. A reversible suppression of protein biosynthesis in Krebs-2 ascites carcinoma cells and wheat germ cell-free systems by CPAs PEG-400, ethylene glycol, glycerol and ME2SO was found. This effect is shown to be stipulated by a direct influence of the studied CPAs on translation processes. ME2SO, glycerol, ethylene glycol and PEG-400 were established to cause the Mg-dependent inhibition of protein biosynthesis in cell-free system of Krebs-2 ascites carcinoma cells in endogenous matrices and wheat germ ones in exogenous matrices. It has been shown that the mechanism of inhibiting action of CPAs on protein biosynthesis in cell-free systems is related to Mg2+-dependent inhibition of tRNA aminoacylation, which when penetrating Me2SO, glycerol and ethylene glycol CPAs are used, has a reversible character, and when PEG-400 being a hardly penetrating CPA is applied it is just partially recovered.

Diadenosine tetraphosphate (Ap(4)A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. Ap(4)A is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap(4)A hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect in vivo levels of Ap(4)A in B. subtilis. YqeK shows a catalytic efficiency toward Ap(4)A similar to that of the symmetrically cleaving Ap(4)A hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals. IMPORTANCE Elevation of Ap(4)A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap(4)A hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap(4)A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae. By identifying the player contributing to Ap(4)A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.

In the Gram-positive Firmicute bacteriumBacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the multi-domain RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we uncover the molecular mechanism of Rel-mediated stringent response. Off the ribosome, Rel assumes a ‘closed’ conformation which has predominantly (p)ppGpp hydrolysis activity. This state does not specifically inspect tRNA and the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and recruitment of cognate deacylated tRNA to the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.

In Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the bifunctional RelA/SpoT Homolog factor Rel, capable of both synthesising and hydrolysing (p)ppGpp. Rel monitors the aminoacylation status of the ribosomal A-site tRNA by inspecting its CCA end. Here we uncover the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity which does not specifically monitor tRNA aminoacylation. Once bound to the ribosome, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for recognition and recruitment of cognate deacylated tRNA to the ribosome. Deacylated tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while hydrolysis is suppressed. The tRNA-dependent locking of Rel on the ribosome and activation of (p)ppGpp synthesis are completely abrogated by tRNA aminoacylation. Binding of pppGpp to an N-terminal allosteric site hyper-enhances its synthetase activity.

The aminoacylation reaction is one of most extensively studied cellular processes. The so-called “canonical” reaction is carried out by direct charging of an amino acid (aa) onto its corresponding transfer RNA (tRNA) by the cognate aminoacyl-tRNA synthetase (aaRS), and the canonical usage of the aminoacylated tRNA (aa-tRNA) is to translate a messenger RNA codon in a translating ribosome. However, four out of the 22 genetically-encoded aa are made “noncanonically” through a two-step or indirect route that usually compensate for a missing aaRS. Additionally, from the 22 proteinogenic aa, 13 are noncanonically used, by serving as substrates for the tRNA- or aa-tRNA-dependent synthesis of other cellular components. These nontranslational processes range from lipid aminoacylation, and heme, aa, antibiotic and peptidoglycan synthesis to protein degradation. This chapter focuses on these noncanonical usages of aa-tRNAs and the ways of generating them, and also highlights the strategies that cells have evolved to balance the use of aa-tRNAs between protein synthesis and synthesis of other cellular components.

International audience; Polypeptides containing β-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of β-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of β3-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds β3-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in β3-Met commercial samples. The 1.45 Å crystal structure of the MetRS: β3-Met complex shows that β3-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved β3-Met aminoacylation capabilities.

With the intense protein synthesis demands of cancer, the classical enzymatic role of aminoacyl-tRNA synthetases (aaRSs) is required to sustain tumor growth. However, many if not all aaRSs also possess regulatory functions outside of the domain of catalytic tRNA aminoacylation, which can further contribute to or even antagonize cancers in non-translational ways. These regulatory functions of aaRS are likely to be manipulated in cancer to ensure uncontrolled growth and survival. This review will largely focus on the unique capacities of individual and sometimes collaborating synthetases to influence the hallmarks of cancer, which represent the principles and characteristics of tumorigenesis. An interesting feature of cytoplasmic aaRSs in higher eukaryotes is the formation of a large multi-synthetase complex (MSC) with nine aaRSs held together by three non-enzymatic scaffolding proteins (AIMPs). The MSC-associated aaRSs, when released from the complex in response to certain stimulations, often participate in pathways that promote tumorigenesis. In contrast, the freestanding aaRSs are associated with activities in both directions-some promoting while others inhibiting cancer. The AIMPs have emerged as potent tumor suppressors through their own distinct mechanisms. We propose that the tumor-suppressive roles of AIMPs may also be a consequence of keeping the cancer-promoting aaRSs within the MSC. The rich connections between cancer and the synthetases have inspired the development of innovative cancer treatments that target or take advantage of these novel functions of aaRSs.