Investigating the Merits of Microfluidic Capillary Zone Electrophoresis-Mass Spectrometry (CZE-MS) in the Bottom-Up Characterization of Larger RNAs.

Established bottom-up approaches for the characterization of nucleic acids (NAs) rely on the strand-cleavage activity of nucleotide-specific endonucleases to generate smaller oligonucleotides amenable to gas-phase sequencing. The complexity of these hydrolytic mixtures calls for the utilization of a front-end separation to facilitate full mass spectrometric (MS) characterization. This report explored the merits of microfluidic capillary zone electrophoresis (CZE) as a possible alternative to common liquid chromatography techniques. An oligonucleotide ladder was initially employed to investigate the roles of fundamental analyte features and experimental parameters in determining the outcome of CZE-MS analyses. The results demonstrated the ability to fully resolve the various rungs into discrete electrophoretic peaks with full-width half-height (FWHH) resolution that was visibly affected by the overall amount of material injected into the system. Analogous results were obtained from a digestion mixture prepared by treating yeast tRNAPhe (75 nt) with RNase T1, which provided several well-resolved peaks in spite of the increasing sample heterogeneity. The regular shapes of such peaks, however, belied the fact that most of them contained sets of comigrating species, as shown by the corresponding MS spectra. Even though it was not possible to segregate each species into an individual electrophoretic peak, the analysis still proved capable of unambiguously identifying a total of 29 hydrolytic products, which were sufficient to cover 96% of the tRNAPhe's sequence. Their masses accurately reflected the presence of modified nucleotides characteristic of this type of substrate. The analysis of a digestion mixture obtained from the 364 nt HIV-1 5'-UTR proved to be more challenging. The electropherogram displayed fewer well-resolved peaks and significantly greater incidence of product comigration. In this case, fractionating the highly heterogeneous mixture into discrete bands helped reduce signal suppression and detection bias. As a result, the corresponding MS data enabled the assignment of 248 products out of the possible 513 predicted from the 5'-UTR sequence, which afforded 100% sequence coverage. This figure represented a significant improvement over the 36 total products identified earlier under suboptimal conditions, which afforded only 57% coverage, or the 83 observed by direct infusion nanospray-MS (72%). These results provided a measure of the excellent potential of the technique to support the bottom-up characterization of progressively larger NA samples, such as putative NA therapeutics and mRNA vaccines.

Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA.

The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Here we identify phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor of the CCCCGG antisense repeat RNA in cytosol. The aminoacylation of tRNAPhe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNAPhe. Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this study reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology.

Clinical and molecular characterization of novel FARS2 variants causing neonatal mitochondrial disease.

FARS2 encodes the mitochondrial phenylalanyl-tRNA synthetase (mtPheRS), which is essential for charging mitochondrial (mt-) tRNAPhe with phenylalanine for use in intramitochondrial translation. Many biallelic, pathogenic FARS2 variants have been described previously, which are mostly associated with two distinct clinical phenotypes; an early onset epileptic mitochondrial encephalomyopathy or a later onset spastic paraplegia. In this study, we report on a patient who presented at 3 weeks of age with tachypnoea and poor feeding, which progressed to severe metabolic decompensation with lactic acidosis and seizure activity followed by death at 9 weeks of age. Rapid trio whole exome sequencing identified compound heterozygous FARS2 variants including a pathogenic exon 2 deletion on one allele and a rare missense variant (c.593G > T, p.(Arg198Leu)) on the other allele, necessitating further work to aid variant classification. Assessment of patient fibroblasts demonstrated severely decreased steady-state levels of mtPheRS, but no obvious defect in any components of the oxidative phosphorylation system. To investigate the potential pathogenicity of the missense variant, we determined its high-resolution crystal structure, demonstrating a local structural destabilization in the catalytic domain. Moreover, the R198L mutation reduced the thermal stability and impaired the enzymatic activity of mtPheRS due to a lower binding affinity for tRNAPhe and a slower turnover rate. Together these data confirm the pathogenicity of this FARS2 variant in causing early-onset mitochondrial epilepsy.

Human SAMD9 is a poxvirus-activatable anticodon nuclease inhibiting codon-specific protein synthesis.

As a defense strategy against viruses or competitors, some microbes use anticodon nucleases (ACNases) to deplete essential tRNAs, effectively halting global protein synthesis. However, this mechanism has not been observed in multicellular eukaryotes. Here, we report that human SAMD9 is an ACNase that specifically cleaves phenylalanine tRNA (tRNAPhe), resulting in codon-specific ribosomal pausing and stress signaling. While SAMD9 ACNase activity is normally latent in cells, it can be activated by poxvirus infection or rendered constitutively active by SAMD9 mutations associated with various human disorders, revealing tRNAPhe depletion as an antiviral mechanism and a pathogenic condition in SAMD9 disorders. We identified the N-terminal effector domain of SAMD9 as the ACNase, with substrate specificity primarily determined by a eukaryotic tRNAPhe-specific 2'-O-methylation at the wobble position, making virtually all eukaryotic tRNAPhe susceptible to SAMD9 cleavage. Notably, the structure and substrate specificity of SAMD9 ACNase differ from known microbial ACNases, suggesting convergent evolution of a common immune defense strategy targeting tRNAs.

A Simple Method for the Detection of Wybutosine-Modified tRNAPheGAA as a Readout of Retrograde tRNA Nuclear Import and Re-export: HCl/Aniline Cleavage and Nonradioactive Northern Hybridization.

tRNAs are highly mobile molecules that are trafficked back and forth between the nucleus and cytoplasm by several proteins. However, characterization of the movement of tRNAs and the proteins mediating these movements can be difficult. Here, we describe an easy and cost-effective assay to discover genes that are involved in two specific tRNA trafficking events, retrograde nuclear import and nuclear re-export for yeast, Saccharomyces cerevisiae. This assay, referred to as the hydrochloric acid (HCl)/aniline assay, identifies the presence or absence of a unique modification on tRNAPheGAA called wybutosine (yW) that requires mature, spliced tRNAPheGAA to undergo retrograde nuclear import and subsequent nuclear re-export for its addition. Therefore, the presence/absence of yW-modified tRNAPheGAA serves as a readout of retrograde nuclear import and nuclear re-export. This simple assay can be used to determine the role of any gene product in these previously elusive tRNA trafficking events.

Insertion of 4-Demethylwyosine in tRNAPhe Catalyzed by the Radical S-Adenosyl-l-methionine Enzyme TYW1 Entails Oxidative Cleavage of Pyruvate to Form CO2.

The radical S-adenosyl-l-methionine (SAM) enzyme TYW1 catalyzes the condensation of C-2 and C-3 atoms of pyruvate with N-methylguanosine containing tRNAPhe to form 4-demethylwyosine (imG-14) modified tRNAPhe. The fate of C-1 is not known, and either formate or carbon dioxide (CO2) has been proposed. In this study, a coupled assay that transforms either CO2 or formate to oxaloacetate (OAA) was used to determine the fate of C-1. In the presence of [1-13C1]-pyruvate, 13C-enriched OAA was observed in a process that is concomitant with the formation of imG-14, under conditions that preferentially transform CO2 and not formate to OAA. These findings are discussed in the context of the cofactor content of TYW1 and a new role for the auxiliary cluster in catalyzing the oxidative cleavage of C-1-C-2 bond of pyruvate in the catalytic cycle of TYW1.

α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain.

The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (α-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that are neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, α-PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated α-PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype partially resembles the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between α-PheRS and Notch suggest that their activities neutralize each other and that elevated α-PheRS levels attenuate Notch signaling when Notch induces differentiation into enterocytes, type II neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of α-PheRS which accumulates naturally in the intestine. This truncated version of α-PheRS (α-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that α-S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in α-PheRS and α-S, but also essential for attenuating Notch signaling.

Heteroplasmic and homoplasmic m.616T>C in mitochondria tRNAPhe promote isolated chronic kidney disease and hyperuricemia.

Inherited kidney diseases are the fifth most common cause of end-stage renal disease (ESRD). Mitochondrial dysfunction plays a vital role in the progression of inherited kidney diseases, while mitochondrial-transfer RNA (mt-tRNA) variants and their pathogenic contributions to kidney disease remain largely unclear. In this study, we identified the pathogenic mt-tRNAPhe 616T>C mutation in 3 families and documented that m.616T>C showed a high pathogenic threshold, with both heteroplasmy and homoplasmy leading to isolated chronic kidney disease and hyperuricemia without hematuria, proteinuria, or renal cyst formation. Moreover, 1 proband with homoplamic m.616T>C presented ESRD as a child. No symptoms of nervous system evolvement were observed in these families. Lymphoblast cells bearing m.616T>C exhibited swollen mitochondria, underwent active mitophagy, and showed respiratory deficiency, leading to reduced mitochondrial ATP production, diminished membrane potential, and overproduction of mitochondrial ROS. Pathogenic m.616T>C abolished a highly conserved base pair (A31-U39) in the anticodon stem-loop which altered the structure of mt-tRNAPhe, as confirmed by a decreased melting temperature and slower electrophoretic mobility of the mutant tRNA. Furthermore, the unstable structure of mt-tRNAPhe contributed to a shortage of steady-state mt-tRNAPhe and enhanced aminoacylation efficiency, which resulted in impaired mitochondrial RNA translation and a significant decrease in mtDNA-encoded polypeptides. Collectively, these findings provide potentially new insights into the pathogenesis underlying inherited kidney disease caused by mitochondrial variants.

Gitelman-Like Syndrome Caused by Pathogenic Variants in mtDNA.

BACKGROUND: Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown. METHODS: We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. RESULTS: Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF), and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. CONCLUSION: Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies.

Mycobacterium tuberculosis Phe-tRNA synthetase: structural insights into tRNA recognition and aminoacylation.

Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3' end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs.

Direct Measurements of Erythromycin's Effect on Protein Synthesis Kinetics in Living Bacterial Cells.

Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNAPhe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNAfMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNAPhe, underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.

Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo.

Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNAPhe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.

The nature of the modification at position 37 of tRNAPhe correlates with acquired taxol resistance.

Acquired drug resistance is a major obstacle in cancer therapy. Recent studies revealed that reprogramming of tRNA modifications modulates cancer survival in response to chemotherapy. However, dynamic changes in tRNA modification were not elucidated. In this study, comparative analysis of the human cancer cell lines and their taxol resistant strains based on tRNA mapping was performed by using UHPLC-MS/MS. It was observed for the first time in all three cell lines that 4-demethylwyosine (imG-14) substitutes for hydroxywybutosine (OHyW) due to tRNA-wybutosine synthesizing enzyme-2 (TYW2) downregulation and becomes the predominant modification at the 37th position of tRNAphe in the taxol-resistant strains. Further analysis indicated that the increase in imG-14 levels is caused by downregulation of TYW2. The time courses of the increase in imG-14 and downregulation of TYW2 are consistent with each other as well as consistent with the time course of the development of taxol-resistance. Knockdown of TYW2 in HeLa cells caused both an accumulation of imG-14 and reduction in taxol potency. Taken together, low expression of TYW2 enzyme promotes the cancer survival and resistance to taxol therapy, implying a novel mechanism for taxol resistance. Reduction of imG-14 deposition offers an underlying rationale to overcome taxol resistance in cancer chemotherapy.

Posttranscriptional modifications at the 37th position in the anticodon stem-loop of tRNA: structural insights from MD simulations.

Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i6A, t6A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m1G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation.

A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae.

In eukaryotes, tRNAs are transcribed in the nucleus and subsequently exported to the cytoplasm where they serve as essential adaptor molecules in translation. However, tRNAs can be returned to the nucleus by the evolutionarily conserved process called tRNA retrograde nuclear import, before relocalization back to the cytoplasm via a nuclear re-export step. Several important functions of these latter two trafficking events have been identified, yet the pathways are largely unknown. Therefore, we developed an assay in Saccharomyces cerevisiae to identify proteins mediating tRNA retrograde nuclear import and re-export using the unique wybutosine modification of mature tRNAPhe. Our hydrochloric acid/aniline assay revealed that the karyopherin Mtr10 mediates retrograde import of tRNAPhe, constitutively and in response to amino acid deprivation, whereas the Hsp70 protein Ssa2 mediates import specifically in the latter. Furthermore, tRNAPhe is re-exported by Crm1 and Mex67, but not by the canonical tRNA exporters Los1 or Msn5. These findings indicate that the re-export process occurs in a tRNA family-specific manner. Together, this assay provides insights into the pathways for tRNAPhe retrograde import and re-export and is a tool that can be used on a genome-wide level to identify additional gene products involved in these tRNA trafficking events.

A novel, mitochondrial, internal tRNA-derived RNA fragment possesses clinical utility as a molecular prognostic biomarker in chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults. The prognosis of CLL patients varies considerably. Transfer RNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNA fragments excised from mature tRNAs and pre-tRNAs located in nuclei as well as in mitochondria. In this study, the clinical utility of i-tRF-PheGAA, a novel mitochondrial tRF, was investigated in CLL. DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 91 CLL patients and 43 non-leukemic controls. Total RNA was isolated from each sample, polyadenylated at the 3' end and reversely transcribed. An in-house developed real-time quantitative PCR assay was developed and applied, and the results were biostatistically analyzed. For the normalization of the i-tRF-PheGAA expression levels, the expression of a small nucleolar RNA (RNU48) was used as reference. RESULTS: Mann-Whitney U test showed that i-tRF-PheGAA can distinguish between CLL samples and normal controls (p < 0.001). As determined by Kaplan-Meier survival analysis, overexpression of i-tRF-PheGAA was related to poor overall survival of the CLL patients (p < 0.001). Univariate bootstrap Cox regression analysis exhibited a higher hazard ratio of 7.95 (95% CI = 2.37-26.72, p < 0.001) for patients with positive i-tRF-PheGAA expression status. Multivariate bootstrap Cox regression analysis showed that the prognostic value of this tRF is independent of clinical stage, mutational status of the immunoglobulin heavy chain variable (IGHV) genetic locus, and CD38 expression status (p = 0.010). CONCLUSIONS: Our results show that i-tRF-PheGAA can serve as a molecular biomarker of poor prognosis in CLL, alongside with the existing factors for CLL prognosis.

1H, 15N chemical shift assignments of the imino groups of yeast tRNAPhe: influence of the post-transcriptional modifications.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In particular, the introduction of post-transcriptional modifications in tRNAs is controlled and influenced by multiple factors. In turn, tRNA biological functions are often modulated by their modifications. Although modifications play essential roles in tRNA biology, methods to directly detect their introduction during tRNA maturation are rare and do not easily provide information on the temporality of modification events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. Here we report the 1H,15N chemical shift assignments of imino groups in three forms of the yeast tRNAPhe differing in their modification content. These assignments are a prerequisite for the time-resolved NMR monitoring of yeast tRNAPhe maturation in yeast extracts.

Breaking a single hydrogen bond in the mitochondrial tRNAPhe -PheRS complex leads to phenotypic pleiotropy of human disease.

Various pathogenic variants in both mitochondrial tRNAPhe and Phenylalanyl-tRNA synthetase mitochondrial protein coding gene (FARS2) gene encoding for the human mitochondrial PheRS have been identified and associated with neurological and/or muscle-related pathologies. An important Guanine-34 (G34)A anticodon mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) syndrome has been reported in hmit-tRNAPhe . The majority of G34 contacts in available aaRSs-tRNAs complexes specifically use that base as an important tRNA identity element. The network of intermolecular interactions providing its specific recognition also largely conserved. However, their conservation depends also on the invariance of the residues in the anticodon binding domain (ABD) of human mitochondrial Phenylalanyl-tRNA synthetase (hmit-PheRS). A defect in recognition of the anticodon of tRNAPhe may happen not only because of G34A mutation, but also due to mutations in the ABD. Indeed, a pathogenic mutation in FARS2 has been recently reported in a 9-year-old female patient harboring a p.Asp364Gly mutation. Asp364 is hydrogen bonded (HB) to G34 in WT hmit-PheRS. Thus, there are two pathogenic variants disrupting HB between G34 and Asp364: one is associated with G34A mutation, and the other with Asp364Gly mutation. We have measured the rates of tRNAPhe aminoacylation catalyzed by WT hmit-PheRS and mutant enzymes. These data ranked the residues making a HB with G34 according to their contribution to activity and the signal transduction pathway in the hmit-PheRS-tRNAPhe complex. Furthermore, we carried out extensive MD simulations to reveal the interdomain contact topology on the dynamic trajectories of the complex, and gaining insight into the structural and dynamic integrity effects of hmit-PheRS complexed with tRNAPhe . DATABASE: Structural data are available in PDB database under the accession number(s): 3CMQ, 3TUP, 5MGH, 5MGV.

Time-resolved NMR monitoring of tRNA maturation.

Although the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. Here, we introduce the application of NMR spectroscopy to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNAPhe with time-resolved NMR measurements, we show that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we show that a strong hierarchy controls the introduction of the T54, Ψ55 and m1A58 modifications in the T-arm, and we demonstrate that the modification circuits identified in yeast extract with NMR also impact the tRNA modification process in living cells. The NMR-based methodology presented here could be adapted to investigate different aspects of tRNA maturation and RNA modifications in general.