tRNA renovatio: Rebirth through fragmentation.

tRNA function is based on unique structures that enable mRNA decoding using anticodon trinucleotides. These structures interact with specific aminoacyl-tRNA synthetases and ribosomes using 3D shape and sequence signatures. Beyond translation, tRNAs serve as versatile signaling molecules interacting with other RNAs and proteins. Through evolutionary processes, tRNA fragmentation emerges as not merely random degradation but an act of recreation, generating specific shorter molecules called tRNA-derived small RNAs (tsRNAs). These tsRNAs exploit their linear sequences and newly arranged 3D structures for unexpected biological functions, epitomizing the tRNA "renovatio" (from Latin, meaning renewal, renovation, and rebirth). Emerging methods to uncover full tRNA/tsRNA sequences and modifications, combined with techniques to study RNA structures and to integrate AI-powered predictions, will enable comprehensive investigations of tRNA fragmentation products and new interaction potentials in relation to their biological functions. We anticipate that these directions will herald a new era for understanding biological complexity and advancing pharmaceutical engineering.

Domain collapse and active site ablation generate a widespread animal mitochondrial seryl-tRNA synthetase.

Through their aminoacylation reactions, aminoacyl tRNA-synthetases (aaRS) establish the rules of the genetic code throughout all of nature. During their long evolution in eukaryotes, additional domains and splice variants were added to what is commonly a homodimeric or monomeric structure. These changes confer orthogonal functions in cellular activities that have recently been uncovered. An unusual exception to the familiar architecture of aaRSs is the heterodimeric metazoan mitochondrial SerRS. In contrast to domain additions or alternative splicing, here we show that heterodimeric metazoan mitochondrial SerRS arose from its homodimeric ancestor not by domain additions, but rather by collapse of an entire domain (in one subunit) and an active site ablation (in the other). The collapse/ablation retains aminoacylation activity while creating a new surface, which is necessary for its orthogonal function. The results highlight a new paradigm for repurposing a member of the ancient tRNA synthetase family.

CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS.

Through dominant mutations, aminoacyl-tRNA synthetases constitute the largest protein family linked to Charcot-Marie-Tooth disease (CMT). An example is CMT subtype 2N (CMT2N), caused by individual mutations spread out in AlaRS, including three in the aminoacylation domain, thereby suggesting a role for a tRNA-charging defect. However, here we found that two are aminoacylation defective but that the most widely distributed R329H is normal as a purified protein in vitro and in unfractionated patient cell samples. Remarkably, in contrast to wild-type (WT) AlaRS, all three mutant proteins gained the ability to interact with neuropilin 1 (Nrp1), the receptor previously linked to CMT pathogenesis in GlyRS. The aberrant AlaRS-Nrp1 interaction is further confirmed in patient samples carrying the R329H mutation. However, CMT2N mutations outside the aminoacylation domain do not induce the Nrp1 interaction. Detailed biochemical and biophysical investigations, including X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter determinations, and protease digestions reveal a mutation-induced structural loosening of the aminoacylation domain that correlates with the Nrp1 interaction. The b1b2 domains of Nrp1 are responsible for the interaction with R329H AlaRS. The results suggest Nrp1 is more broadly associated with CMT-associated members of the tRNA synthetase family. Moreover, we revealed a distinct structural loosening effect induced by a mutation in the editing domain and a lack of conformational impact with C-Ala domain mutations, indicating mutations in the same protein may cause neuropathy through different mechanisms. Our results show that, as with other CMT-associated tRNA synthetases, aminoacylation per se is not relevant to the 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.

Cross-editing by a tRNA synthetase allows vertebrates to abundantly express mischargeable tRNA without causing mistranslation.

The accuracy in pairing tRNAs with correct amino acids by aminoacyl-tRNA synthetases (aaRSs) dictates the fidelity of translation. To ensure fidelity, multiple aaRSs developed editing functions that remove a wrong amino acid from tRNA before it reaches the ribosome. However, no specific mechanism within an aaRS is known to handle the scenario where a cognate amino acid is mischarged onto a wrong tRNA, as exemplified by AlaRS mischarging alanine to G4:U69-containing tRNAThr. Here, we report that the mischargeable G4:U69-containing tRNAThr are strictly conserved in vertebrates and are ubiquitously and abundantly expressed in mammalian cells and tissues. Although these tRNAs are efficiently mischarged, no corresponding Thr-to-Ala mistranslation is detectable. Mistranslation is prevented by a robust proofreading activity of ThrRS towards Ala-tRNAThr. Therefore, while wrong amino acids are corrected within an aaRS, a wrong tRNA is handled in trans by an aaRS cognate to the mischarged tRNA species. Interestingly, although Ala-tRNAThr mischarging is not known to occur in bacteria, Escherichia coli ThrRS also possesses robust cross-editing ability. We propose that the cross-editing activity of ThrRS is evolutionarily conserved and that this intrinsic activity allows G4:U69-containing tRNAThr to emerge and be preserved in vertebrates to have alternative functions without compromising translational fidelity.

CMT disease severity correlates with mutation-induced open conformation of histidyl-tRNA synthetase, not aminoacylation loss, in patient cells.

Aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are the largest protein family causatively linked to neurodegenerative Charcot-Marie-Tooth (CMT) disease. Dominant mutations cause the disease, and studies of CMT disease-causing mutant glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase (TyrRS) showed their mutations create neomorphic structures consistent with a gain-of-function mechanism. In contrast, based on a haploid yeast model, loss of aminoacylation function was reported for CMT disease mutants in histidyl-tRNA synthetase (HisRS). However, neither that nor prior work of any CMT disease-causing aaRS investigated the aminoacylation status of tRNAs in the cellular milieu of actual patients. Using an assay that interrogated aminoacylation levels in patient cells, we investigated a HisRS-linked CMT disease family with the most severe disease phenotype. Strikingly, no difference in charged tRNA levels between normal and diseased family members was found. In confirmation, recombinant versions of 4 other HisRS CMT disease-causing mutants showed no correlation between activity loss in vitro and severity of phenotype in vivo. Indeed, a mutation having the most detrimental impact on activity was associated with a mild disease phenotype. In further work, using 3 independent biophysical analyses, structural opening (relaxation) of mutant HisRSs at the dimer interface best correlated with disease severity. In fact, the HisRS mutation in the severely afflicted patient family caused the largest degree of structural relaxation. These data suggest that HisRS-linked CMT disease arises from open conformation-induced mechanisms distinct from loss of aminoacylation.

Distinct ways of G:U recognition by conserved tRNA binding motifs.

Throughout three domains of life, alanyl-tRNA synthetases (AlaRSs) recognize a G3:U70 base pair in the acceptor stem of tRNAAla as the major identity determinant of tRNAAla The crystal structure of the archaeon Archaeoglobus fulgidus AlaRS in complex with tRNAAla provided the basis for G3:U70 recognition with residues (Asp and Asn) that are conserved in the three domains [Naganuma M, et al. (2014) Nature 510:507-511]. The recognition mode is unprecedented, with specific accommodation of the dyad asymmetry of the G:U wobble pair and exclusion of the dyad symmetry of a Watson-Crick pair. With this conserved mode, specificity is based more on "fit" than on direct recognition of specific atomic groups. Here, we show that, in contrast to the archaeal complex, the Escherichia coli enzyme uses direct positive (energetically favorable) minor groove recognition of the unpaired 2-amino of G3 by Asp and repulsion of a competing base pair by Asn. Strikingly, mutations that disrupted positive recognition by the E. coli enzyme had little or no effect on G:U recognition by the human enzyme. Alternatively, Homo sapiens AlaRS selects G:U without positive recognition and uses Asp instead to repel a competitor. Thus, the widely conserved Asp-plus-Asn architecture of AlaRSs can select G:U in a straightforward (bacteria) or two different unconventional (eukarya/archaea) ways. The adoption of different modes for recognition of a widely conserved G:U pair in alanine tRNAs suggests an early and insistent role for G:U in the development of the genetic code.

A monovalent cation acts as structural and catalytic cofactor in translational GTPases.

Translational GTPases are universally conserved GTP hydrolyzing enzymes, critical for fidelity and speed of ribosomal protein biosynthesis. Despite their central roles, the mechanisms of GTP-dependent conformational switching and GTP hydrolysis that govern the function of trGTPases remain poorly understood. Here, we provide biochemical and high-resolution structural evidence that eIF5B and aEF1A/EF-Tu bound to GTP or GTPγS coordinate a monovalent cation (M(+)) in their active site. Our data reveal that M(+) ions form constitutive components of the catalytic machinery in trGTPases acting as structural cofactor to stabilize the GTP-bound "on" state. Additionally, the M(+) ion provides a positive charge into the active site analogous to the arginine-finger in the Ras-RasGAP system indicating a similar role as catalytic element that stabilizes the transition state of the hydrolysis reaction. In sequence and structure, the coordination shell for the M(+) ion is, with exception of eIF2γ, highly conserved among trGTPases from bacteria to human. We therefore propose a universal mechanism of M(+)-dependent conformational switching and GTP hydrolysis among trGTPases with important consequences for the interpretation of available biochemical and structural data.

eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining.

eIF5B is a eukaryal translational GTPase that catalyzes ribosomal subunit joining to form elongation-competent ribosomes. Despite its central role in protein synthesis, the mechanistic details that govern the function of eIF5B or its archaeal and bacterial (IF2) orthologs remained unclear. Here, we present six high-resolution crystal structures of eIF5B in its apo, GDP- and GTP-bound form that, together with an analysis of the thermodynamics of nucleotide binding, provide a detailed picture of the entire nucleotide cycle performed by eIF5B. Our data show that GTP binding induces significant conformational changes in the two conserved switch regions of the G domain, resulting in the reorganization of the GTPase center. These rearrangements are accompanied by the rotation of domain II relative to the G domain and release of domain III from its stable contacts with switch 2, causing an increased intrinsic flexibility in the free GTP-bound eIF5B. Based on these data, we propose a novel domain release mechanism for eIF5B/IF2 activation that explains how eIF5B and IF2 fulfill their catalytic role during ribosomal subunit joining.

Architecture of the eIF2B regulatory subcomplex and its implications for the regulation of guanine nucleotide exchange on eIF2.

Eukaryal translation initiation factor 2B (eIF2B) acts as guanine nucleotide exchange factor (GEF) for eIF2 and forms a central target for pathways regulating global protein synthesis. eIF2B consists of five non-identical subunits (α-ϵ), which assemble into a catalytic subcomplex (γ, ϵ) responsible for the GEF activity, and a regulatory subcomplex (α, ß, δ) which regulates the GEF activity under stress conditions. Here, we provide new structural and functional insight into the regulatory subcomplex of eIF2B (eIF2B(RSC)). We report the crystal structures of eIF2Bß and eIF2Bδ from Chaetomium thermophilum as well as the crystal structure of their tetrameric eIF2B(ßδ)2 complex. Combined with mutational and biochemical data, we show that eIF2B(RSC) exists as a hexamer in solution, consisting of two eIF2Bßδ heterodimers and one eIF2Bα2 homodimer, which is homologous to homohexameric ribose 1,5-bisphosphate isomerases. This homology is further substantiated by the finding that eIF2Bα specifically binds AMP and GMP as ligands. Based on our data, we propose a model for eIF2B(RSC) and its interactions with eIF2 that is consistent with previous biochemical and genetic data and provides a framework to better understand eIF2B function, the molecular basis for Gcn(-), Gcd(-) and VWM/CACH mutations and the evolutionary history of the eIF2B complex.

Structural insight into the recognition of amino-acylated initiator tRNA by eIF5B in the 80S initiation complex.

BACKGROUND: From bacteria to eukarya, the specific recognition of the amino-acylated initiator tRNA by the universally conserved translational GTPase eIF5B/IF2 is one of the most central interactions in the process of translation initiation. However, the molecular details, particularly also in the context of ribosomal initiation complexes, are only partially understood. RESULTS: A reinterpretation of the 6.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the eukaryal 80S initiation complex using the recently published crystal structure of eIF5B reveals that domain IV of eIF5B forms extensive interaction interfaces with the Met-tRNAi, which, in contrast to the previous model, directly involve the methionylated 3' CCA-end of the acceptor stem. These contacts are mediated by a conserved surface area, which is homologous to the surface areas mediating the interactions between IF2 and fMet-tRNAfMet as well as between domain II of EF-Tu and amino-acylated elongator tRNAs. CONCLUSIONS: The reported observations provide novel direct structural insight into the specific recognition of the methionylated acceptor stem by eIF5B domain IV and demonstrate its universality among eIF5B/IF2 orthologs in the three domains of life.

Molecular basis for the interactions of eIF2ß with eIF5, eIF2B, and 5MP1 and their regulation by CK2.

The heterotrimeric GTPase eukaryotic translation initiation factor 2 (eIF2) delivers the initiator Met-tRNAi to the ribosomal translation preinitiation complex. eIF2ß has three lysine-rich repeats (K-boxes) in its N-terminal tail, which are important for binding to the GTPase-activating protein (GAP) eIF5, the guanine nucleotide exchange factor (GEF) eIF2B, and the regulator eIF5-mimic protein (5MP). Here, we combine X-ray crystallography with NMR to understand the molecular basis and dynamics of these interactions. The crystal structure of yeast eIF5-CTD in complex with K-box 3 of eIF2ß reveals an extended binding site on eIF2ß, far beyond the K-box. We show that human eIF5, eIF2Bε, and 5MP1 can all bind to each of the three K-boxes, while reducing each other's affinities. Moreover, all these affinities are increased by CK2 phosphomimetic mutations. Our results reveal how eIF5, eIF2B, and 5MP displace each other from eIF2, and elucidate the role of CK2 in remodeling the translation apparatus.

Structural basis for a degenerate tRNA identity code and the evolution of bimodal specificity in human mitochondrial tRNA recognition.

Animal mitochondrial gene expression relies on specific interactions between nuclear-encoded aminoacyl-tRNA synthetases and mitochondria-encoded tRNAs. Their evolution involves an antagonistic interplay between strong mutation pressure on mtRNAs and selection pressure to maintain their essential function. To understand the molecular consequences of this interplay, we analyze the human mitochondrial serylation system, in which one synthetase charges two highly divergent mtRNASer isoacceptors. We present the cryo-EM structure of human mSerRS in complex with mtRNASer(UGA), and perform a structural and functional comparison with the mSerRS-mtRNASer(GCU) complex. We find that despite their common function, mtRNASer(UGA) and mtRNASer(GCU) show no constrain to converge on shared structural or sequence identity motifs for recognition by mSerRS. Instead, mSerRS evolved a bimodal readout mechanism, whereby a single protein surface recognizes degenerate identity features specific to each mtRNASer. Our results show how the mutational erosion of mtRNAs drove a remarkable innovation of intermolecular specificity rules, with multiple evolutionary pathways leading to functionally equivalent outcomes.

Structural basis for shape-selective recognition and aminoacylation of a D-armless human mitochondrial tRNA.

Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNASer(GCU). These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNASer(GCU) is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNASer(GCU) evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.

Neuropilin 1 and its inhibitory ligand mini-tryptophanyl-tRNA synthetase inversely regulate VE-cadherin turnover and vascular permeability.

The formation of a functional blood vessel network relies on the ability of endothelial cells (ECs) to dynamically rearrange their adhesive contacts in response to blood flow and guidance cues, such as vascular endothelial growth factor-A (VEGF-A) and class 3 semaphorins (SEMA3s). Neuropilin 1 (NRP1) is essential for blood vessel development, independently of its ligands VEGF-A and SEMA3, through poorly understood mechanisms. Grounding on unbiased proteomic analysis, we report here that NRP1 acts as an endocytic chaperone primarily for adhesion receptors on the surface of unstimulated ECs. NRP1 localizes at adherens junctions (AJs) where, interacting with VE-cadherin, promotes its basal internalization-dependent turnover and favors vascular permeability initiated by histamine in both cultured ECs and mice. We identify a splice variant of tryptophanyl-tRNA synthetase (mini-WARS) as an unconventionally secreted extracellular inhibitory ligand of NRP1 that, by stabilizing it at the AJs, slows down both VE-cadherin turnover and histamine-elicited endothelial leakage. Thus, our work shows a role for NRP1 as a major regulator of AJs plasticity and reveals how mini-WARS acts as a physiological NRP1 inhibitory ligand in the control of VE-cadherin endocytic turnover and vascular permeability.

PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications.

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs-mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)-that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.

Author Correction: PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications.

A Correction to this paper has been published: https://doi.org/10.1038/s41556-021-00687-w.

Relaxed sequence constraints favor mutational freedom in idiosyncratic metazoan mitochondrial tRNAs.

Metazoan complexity and life-style depend on the bioenergetic potential of mitochondria. However, higher aerobic activity and genetic drift impose strong mutation pressure and risk of irreversible fitness decline in mitochondrial (mt)DNA-encoded genes. Bilaterian mitochondria-encoded tRNA genes, key players in mitochondrial activity, have accumulated mutations at significantly higher rates than their cytoplasmic counterparts, resulting in foreshortened and fragile structures. Here we show that fragility of mt tRNAs coincided with the evolution of bilaterian animals. We demonstrate that bilaterians compensated for this reduced structural complexity in mt tRNAs by sequence-independent induced-fit adaption to the cognate mitochondrial aminoacyl-tRNA synthetase (aaRS). Structural readout by nuclear-encoded aaRS partners relaxed the sequence constraints on mt tRNAs and facilitated accommodation of functionally disruptive mutational insults by cis-acting epistatic compensations. Our results thus suggest that mutational freedom in mt tRNA genes is an adaptation to increased mutation pressure that was associated with the evolution of animal complexity.

Translation initiation factor eIF3b contains a nine-bladed ß-propeller and interacts with the 40S ribosomal subunit.

The multisubunit eukaryotic translation initiation factor 3, among which the subunit b (eIF3b) is a major scaffold protein, plays essential roles in protein synthesis. Here, we report the crystal structure of the WD40 domain of Chaetomium thermophilum eIF3b, revealing a nine-bladed ß-propeller fold. Sequence analysis indicates that this propeller architecture is common to all eIF3b orthologs. Revisiting the cryoelectron microscopy (cryo-EM) map of the 43S preinitiation complex suggests an interaction of the eIF3b with the 40S ribosomal subunit involving the ribosomal protein S9e and the 18S rRNA. This model is strongly supported by the direct binding of eIF3b to 40S ribosomes and to the isolated ribosomal protein rpS9e in vitro.