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.

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.

Improved application of RNAModMapper - An RNA modification mapping software tool - For analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data.

Research into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples. RNAModMapper was recently developed as a tool to improve the interpretation and annotation of LC-MS/MS data sets from samples containing post-transcriptionally modified RNAs. Here, we delve deeper into the methodology and practice of RNAModMapper to provide greater insight into its utility, and remaining hurdles, in current RNA modification mapping experiments.

Molecular Mechanism for Folding Cooperativity of Functional RNAs in Living Organisms.

A diverse set of organisms has adapted to live under extreme conditions. The molecular origin of the stability is unclear, however. It is not known whether the adaptation of functional RNAs, which have intricate tertiary structures, arises from strengthening of tertiary or secondary structure. Herein we evaluate effects of sequence changes on the thermostability of tRNAphe using experimental and computational approaches. To separate out effects of secondary and tertiary structure on thermostability, we modify base pairing strength in the acceptor stem, which does not participate in tertiary structure. In dilute solution conditions, strengthening secondary structure leads to non-two-state thermal denaturation curves and has small effects on thermostability, or the temperature at which tertiary structure and function are lost. In contrast, under cellular conditions with crowding and Mg2+-chelated amino acids, where two-state cooperative unfolding is maintained, strengthening secondary structure enhances thermostability. Investigation of stabilities of each tRNA stem across 44 organisms with a range of optimal growing temperatures revealed that organisms that grow in warmer environments have more stable stems. We also used Shannon entropies to identify positions of higher and lower information content, or sequence conservation, in tRNAphe and found that secondary structures have modest information content allowing them to drive thermal adaptation, while tertiary structures have maximal information content hindering them from participating in thermal adaptation. Base-paired regions with no tertiary structure and modest information content thus offer a facile evolutionary route to enhancing the thermostability of functional RNA by the simple molecular rules of base pairing.

Ruthenium anticancer agent KP1019 binds more tightly than NAMI-A to tRNAPhe.

The ruthenium-based anticancer agent NAMI-A (ImH[trans-RuCl4(dmso)(Im)], where Im = imidazole) has been shown to interact with RNA in vivo and in vitro. We hypothesized that the similarly structured drug KP1019 (IndH[trans-RuCl4(Ind)2], where Ind = indazole) binds to RNA as well. Fluorescence spectroscopy was employed to assay the interactions between either NAMI-A or KP1019 and tRNAPhe through an intrinsic fluorophore wybutosine (Y) base and by extrinsic displacement of the intercalating agent ethidium bromide. In both the intrinsic Y-base and extrinsic ethidium bromide studies, KP1019 exhibited tighter binding to phenylalanine-specific tRNA (tRNAPhe) than NAMI-A. In the ethidium bromide study, reducing both drugs from RuIII to RuII resulted in a significant decrease in binding. Our findings suggest that the relatively large heteroaromatic indazole ligands of KP1019 intercalate in the π-stacks of tRNAPhe within structurally complex binding pockets. In addition, NAMI-A appears to be sensitive to destabilizing electrostatic interactions with the negative phosphate backbone of tRNAPhe. Interactions with additional tRNA molecules and other types of RNA require further evaluation to determine the role of RNA in the mechanisms of action for KP1019 and to better understand how Ru drugs fundamentally interact with biomolecules that are more structurally sophisticated than short DNA oligonucleotides. To the best of our knowledge, this is the first study to report KP1019 binding interactions with RNA.

Conformational preferences and structural analysis of hypermodified nucleoside, peroxywybutosine (o2yW) found at 37th position in anticodon loop of tRNAPhe and its role in modulating UUC codon-anticodon interactions.

Hypermodified bases present at 3'-adjacent (37th) position in anticodon loop of tRNAPhe are well known for their contribution in modulating codon-anticodon interactions. Peroxywybutosine (o2yW), a wyosine family member, is one of such tricyclic modified bases observed at the 37th position in tRNAPhe. Conformational preferences and three-dimensional structural analysis of peroxywybutosine have not been investigated in detail at atomic level. Hence, in the present study quantum chemical semi-empirical RM1 and multiple molecular dynamics (MD) simulations have been used to study structural significance of peroxywybutosine in tRNAPhe. Full geometry optimizations over the peroxywybutosine base have also been performed using ab-initio HF-SCF (6-31G**), DFT (B3LYP/6-31G**) and semi-empirical PM6 method to compare the salient properties. RM1 predicted most stable structure shows that the amino-carboxy-propyl side chain of o2yW remains 'distal' to the five membered imidazole ring of tricyclic guanosine. MD simulation trajectory of the isolated peroxy base showed restricted periodical fluctuations of peroxywybutosine side chain which might be helpful to maintain proper anticodon loop structure and mRNA reading frame during protein biosynthesis process. Another comparative MD simulation study of the anticodon stem loop with codon UUC showed various properties, which justify the functional implications of peroxywybutosine at 37th position along with other modified bases present in ASL of tRNAPhe. Thus, this study presents an atomic view into the structural properties of peroxywybutosine, which can be useful to determine its role in the anticodon stem loop in context of codon-anticodon interactions and frame shift mutations.

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis.

Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline.

Cooperative RNA Folding under Cellular Conditions Arises From Both Tertiary Structure Stabilization and Secondary Structure Destabilization.

RNA folding has been studied extensively in vitro, typically under dilute solution conditions and abiologically high salt concentrations of 1 M Na+ or 10 mM Mg2+. The cellular environment is very different, with 20-40% crowding and only 10-40 mM Na+, 140 mM K+, and 0.5-2.0 mM Mg2+. As such, RNA structures and functions can be radically altered under cellular conditions. We previously reported that tRNAphe secondary and tertiary structures unfold together in a cooperative two-state fashion under crowded in vivo-like ionic conditions, but in a noncooperative multistate fashion under dilute in vitro ionic conditions unless in nonphysiologically high concentrations of Mg2+. The mechanistic basis behind these effects remains unclear, however. To address the mechanism that drives RNA folding cooperativity, we probe effects of cellular conditions on structures and stabilities of individual secondary structure fragments comprising the full-length RNA. We elucidate effects of a diverse set of crowders on tRNA secondary structural fragments and full-length tRNA at three levels: at the nucleotide level by temperature-dependent in-line probing, at the tertiary structure level by small-angle X-ray scattering, and at the global level by thermal denaturation. We conclude that cooperative RNA folding is induced by two overlapping mechanisms: increased stability and compaction of tertiary structure through effects of Mg2+, and decreased stability of certain secondary structure elements through the effects of molecular crowders. These findings reveal that despite having very different chemical makeups RNA and protein can both have weak secondary structures in vivo leading to cooperative folding.

Kinetic and structural changes in HsmtPheRS, induced by pathogenic mutations in human FARS2.

Mutations in the mitochondrial aminoacyl-tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS. Patient symptoms range from spastic paraplegia to fatal infantile Alpers encephalopathy. How clinical manifestations of these mutations relate to the changes in three-dimensional structures and kinetic characteristics remains unclear, although impaired aminoacylation has been proposed as possible etiology of diseases. Here, we report four crystal structures of HsmtPheRS mutants, and extensive MD simulations for wild-type and nine mutants to reveal the structural changes on dynamic trajectories of HsmtPheRS. Using steady-state kinetic measurements of phenylalanine activation and tRNAPhe aminoacylation, we gained insight into the structural and kinetic effects of mitochondrial disease-related mutations in FARS2 gene.