tRNA Dysregulation in Neurodevelopmental and Neurodegenerative Diseases.

Transfer RNAs (tRNAs) decode messenger RNA codons to peptides at the ribosome. The nuclear genome contains many tRNA genes for each amino acid and even each anticodon. Recent evidence indicates that expression of these tRNAs in neurons is regulated, and they are not functionally redundant. When specific tRNA genes are nonfunctional, this results in an imbalance between codon demand and tRNA availability. Furthermore, tRNAs are spliced, processed, and posttranscriptionally modified. Defects in these processes lead to neurological disorders. Finally, mutations in the aminoacyl tRNA synthetases (aaRSs) also lead to disease. Recessive mutations in several aaRSs cause syndromic disorders, while dominant mutations in a subset of aaRSs lead to peripheral neuropathy, again due to an imbalance between tRNA supply and codon demand. While it is clear that disrupting tRNA biology often leads to neurological disease, additional research is needed to understand the sensitivity of neurons to these changes.

Expanding and reprogramming the genetic code of cells and animals.

Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.

Mitochondrial Threonyl-tRNA Synthetase TARS2 Is Required for Threonine-Sensitive mTORC1 Activation.

Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.

Stresses that Raise Np4A Levels Induce Protective Nucleoside Tetraphosphate Capping of Bacterial RNA.

Present in all realms of life, dinucleoside tetraphosphates (Np4Ns) are generally considered signaling molecules. However, only a single pathway for Np4N signaling has been delineated in eukaryotes, and no receptor that mediates the influence of Np4Ns has ever been identified in bacteria. Here we show that, under disulfide stress conditions that elevate cellular Np4N concentrations, diverse Escherichia coli mRNAs and sRNAs acquire a cognate Np4 cap. Purified E. coli RNA polymerase and lysyl-tRNA synthetase are both capable of adding such 5' caps. Cap removal by either of two pyrophosphatases, ApaH or RppH, triggers rapid RNA degradation in E. coli. ApaH, the predominant decapping enzyme, functions as both a sensor and an effector of disulfide stress, which inactivates it. These findings suggest that the physiological changes attributed to elevated Np4N concentrations in bacteria may result from widespread Np4 capping, leading to altered RNA stability and consequent changes in gene expression.

AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells.

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

Mitochondrial aminoacyl-tRNA synthetases trigger unique compensatory mechanisms in neurons.

Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.

tRNAVal allows four-way decoding with unmodified uridine at the wobble position in Lactobacillus casei.

Modifications at the wobble position (position 34) of tRNA facilitate interactions that enable or stabilize non-Watson-Crick basepairs. In bacterial tRNA, 5-hydroxyuridine (ho5U) derivatives xo5U [x: methyl (mo5U), carboxymethyl (cmo5U), and methoxycarbonylmethyl (mcmo5U)] present at the wobble positions of tRNAs are responsible for recognition of NYN codon families. These modifications of U34 allow basepairing not only with A and G but also with U and in some cases C. mo5U was originally found in Gram-positive bacteria, and cmo5U and mcmo5U were found in Gram-negative bacteria. tRNAs of Mycoplasma species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. Lactobacillus casei, Gram-positive bacteria and lactic acid bacteria, lacks the modification enzyme genes for xo5U biosynthesis. Nevertheless, L. casei has only one type of tRNAVal with the anticodon UAC [tRNAVal(UAC)]. However, the genome of L. casei encodes an undetermined tRNA (tRNAUnd) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in L. casei tRNAVal is unmodified and that there is no tRNAUnd expression in the cells. In addition, in vitro transcribed tRNAUnd was not aminoacylated by L. casei valyl-tRNA synthetase suggesting that tRNAUnd is not able to accept valine, even if expressed in cells. Correspondingly, native tRNAVal(UAC) with unmodified U34 bound to all four valine codons in the ribosome A site. This suggests that L. casei tRNAVal decodes all valine codons by four-way decoding, similarly to tRNAs from Mycoplasma species, mitochondria, and chloroplasts.

Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy.

Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by Tars2), disruption of which accumulated Ser-tRNAThr and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.

Murine nuclear tyrosyl-tRNA synthetase deficiency leads to fat storage deficiency and hearing loss.

Aminoacyl-tRNA synthetases are fundamental to the translation machinery with emerging roles in transcriptional regulation. Previous cellular studies have demonstrated tyrosyl-tRNA synthetase (YARS1 or TyrRS) as a stress response protein through its cytosol-nucleus translocation to maintain cellular homeostasis. Here, we established a mouse model with a disrupted TyrRS nuclear localization signal, revealing its systemic impact on metabolism. Nuclear TyrRS deficiency (YarsΔNLS) led to reduced lean mass, reflecting a mild developmental defect, and reduced fat mass, possibly due to increased energy expenditure. Consistently, YarsΔNLS mice exhibit improved insulin sensitivity and reduced insulin levels, yet maintain normoglycemia, indicative of enhanced insulin action. Notably, YarsΔNLS mice also develop progressive hearing loss. These findings underscore the crucial function of nuclear TyrRS in the maintenance of fat storage and hearing and suggest that aminoacyl-tRNA synthetases' regulatory roles can affect metabolic pathways and tissue-specific health. This work broadens our understanding of how protein synthesis interconnects metabolic regulation to ensure energy efficiency.

Factors governing the transcriptome changes and chronological lifespan of fission yeast during phosphate starvation.

Starvation of Schizosaccharomyces pombe for inorganic phosphate elicits adaptive transcriptome changes in which mRNAs driving ribosome biogenesis, tRNA biogenesis, and translation are globally downregulated, while those for autophagy and phosphate mobilization are upregulated. Here, we interrogated three components of the starvation response: upregulated autophagy; the role of transcription factor Pho7 (an activator of the PHO regulon); and upregulated expression of ecl3, one of three paralogous genes (ecl1, ecl2, and ecl3) collectively implicated in cell survival during other nutrient stresses. Ablation of autophagy factor Atg1 resulted in early demise of phosphate-starved fission yeast, as did ablation of Pho7. Transcriptome profiling of phosphate-starved pho7Δ cells highlighted Pho7 as an activator of genes involved in phosphate acquisition and mobilization, not limited to the original three-gene PHO regulon, and additional starvation-induced genes (including ecl3) not connected to phosphate dynamics. Pho7-dependent gene induction during phosphate starvation tracked with the presence of Pho7 DNA-binding elements in the gene promoter regions. Fewer ribosome protein genes were downregulated in phosphate-starved pho7Δ cells versus WT, which might contribute to their shortened lifespan. An ecl3Δ mutant elicited no gene expression changes in phosphate-replete cells and had no impact on survival during phosphate starvation. By contrast, pan-ecl deletion (ecl123Δ) curtailed lifespan during chronic phosphate starvation. Phosphate-starved ecl123Δ cells experienced a more widespread downregulation of mRNAs encoding aminoacyl tRNA synthetases vis-à-vis WT or pho7Δ cells. Collectively, these results enhance our understanding of fission yeast phosphate homeostasis and survival during nutrient deprivation.

RNA modifying enzymes shape tRNA biogenesis and function.

Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with respect to the extraordinary diversity in tRNA modification chemistry. However, the functions of many different tRNA modifications are only beginning to emerge. tRNAs have two general clusters of modifications. The first cluster is within the anticodon stem-loop including several modifications essential for protein translation. The second cluster of modifications is within the tRNA elbow, and roles for these modifications are less clear. In general, tRNA elbow modifications are typically not essential for cell growth, but nonetheless several tRNA elbow modifications have been highly conserved throughout all domains of life. In addition to forming modifications, many tRNA modifying enzymes have been demonstrated or hypothesized to also play an important role in folding tRNA acting as tRNA chaperones. In this review, we summarize the known functions of tRNA modifying enzymes throughout the lifecycle of a tRNA molecule, from transcription to degradation. Thereby, we describe how tRNA modification and folding by tRNA modifying enzymes enhance tRNA maturation, tRNA aminoacylation, and tRNA function during protein synthesis, ultimately impacting cellular phenotypes and disease.

Complex Genomes of Early Nucleocytoviruses Revealed by Ancient Origins of Viral Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (aaRSs), also known as tRNA ligases, are essential enzymes in translation. Owing to their functional essentiality, these enzymes are conserved in all domains of life and used as informative markers to trace the evolutionary history of cellular organisms. Unlike cellular organisms, viruses generally lack aaRSs because of their obligate parasitic nature, but several large and giant DNA viruses in the phylum Nucleocytoviricota encode aaRSs in their genomes. The discovery of viral aaRSs led to the idea that the phylogenetic analysis of aaRSs can shed light on ancient viral evolution. However, conflicting results have been reported from previous phylogenetic studies: one posited that nucleocytoviruses recently acquired their aaRSs from their host eukaryotes, while another hypothesized that the viral aaRSs have ancient origins. Here, we investigated 4,168 nucleocytovirus genomes, including metagenome-assembled genomes (MAGs) derived from large-scale metagenomic studies. In total, we identified 780 viral aaRS sequences in 273 viral genomes. We generated and examined phylogenetic trees of these aaRSs with a large set of cellular sequences to trace evolutionary relationships between viral and cellular aaRSs. The analyses suggest that the origins of some viral aaRSs predate the last common eukaryotic ancestor. Inside viral aaRS clades, we identify intricate evolutionary trajectories of viral aaRSs with horizontal transfers, losses, and displacements. Overall, these results suggest that ancestral nucleocytoviruses already developed complex genomes with an expanded set of aaRSs in the proto-eukaryotic era.

Switch of ELF3 and ATF4 transcriptional axis programs the amino acid insufficiency-linked epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) that endows cancer cells with increased invasive and migratory capacity enables cancer dissemination and metastasis. This process is tightly associated with metabolic reprogramming acquired for rewiring cell status and signaling pathways for survival in dietary insufficiency conditions. However, it remains largely unclear how transcription factor (TF)-mediated transcriptional programs are modulated during the EMT process. Here, we reveal that depletion of a key epithelial TF, ELF3 (E74-like factor-3), triggers a transforming growth factor ß (TGF-ß) signaling activation-like mesenchymal transcriptomic profile and metastatic features linked to the aminoacyl-tRNA biogenesis pathway. Moreover, the transcriptome alterations elicited by ELF3 depletion perfectly resemble an ATF4-dependent weak response to amino acid starvation. Intriguingly, we observe an exclusive enrichment of ELF3 and ATF4 in epithelial and TGF-ß-induced or ELF3-depletion-elicited mesenchymal enhancers, respectively, with rare co-binding on altered enhancers. We also find that the upregulation of aminoacyl-tRNA synthetases and some mesenchymal genes upon amino acid deprivation is diminished in ATF4-depleted cells. In sum, the loss of ELF3 binding on epithelial enhancers and the gain of ATF4 binding on the enhancers of mesenchymal factors and amino acid deprivation responsive genes facilitate the loss of epithelial cell features and the gain of TGF-ß-signaling-associated mesenchymal signatures, which further promote lung cancer cell metastasis.

Multimodal cotranslational interactions direct assembly of the human multi-tRNA synthetase complex.

Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)-essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the ∼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a "multisite" mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second "piggy-back" mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.

Loss of threonyl-tRNA synthetase-like protein Tarsl2 has little impact on protein synthesis but affects mouse development.

Aminoacyl-tRNA synthetases (aaRSs) are essential components for mRNA translation. Two sets of aaRSs are required for cytoplasmic and mitochondrial translation in vertebrates. Interestingly, TARSL2 is a recently evolved duplicated gene of TARS1 (encoding cytoplasmic threonyl-tRNA synthetase) and represents the only duplicated aaRS gene in vertebrates. Although TARSL2 retains the canonical aminoacylation and editing activities in vitro, whether it is a true tRNA synthetase for mRNA translation in vivo is unclear. In this study, we showed that Tars1 is an essential gene since homozygous Tars1 KO mice were lethal. In contrast, when Tarsl2 was deleted in mice and zebrafish, neither the abundance nor the charging levels of tRNAThrs were changed, indicating that cells relied on Tars1 but not on Tarsl2 for mRNA translation. Furthermore, Tarsl2 deletion did not influence the integrity of the multiple tRNA synthetase complex, suggesting that Tarsl2 is a peripheral member of the multiple tRNA synthetase complex. Finally, we observed that Tarsl2-deleted mice exhibited severe developmental retardation, elevated metabolic capacity, and abnormal bone and muscle development after 3 weeks. Collectively, these data suggest that, despite its intrinsic activity, loss of Tarsl2 has little influence on protein synthesis but does affect mouse development.

Sequence-specific targeting of Caenorhabditis elegans C-Ala to the D-loop of tRNAAla.

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.

Mistranslation of the genetic code by a new family of bacterial transfer RNAs.

The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or posttranslational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and support the concept that mistranslation is a mechanism for cellular resiliency against environmental stress.

Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Rewiring of Aminoacyl-tRNA Synthetase Localization and Interactions in Plants With Extensive Mitochondrial tRNA Gene Loss.

The number of tRNAs encoded in plant mitochondrial genomes varies considerably. Ongoing loss of bacterial-like mitochondrial tRNA genes in many lineages necessitates the import of nuclear-encoded counterparts that share little sequence similarity. Because tRNAs are involved in highly specific molecular interactions, this replacement process raises questions about the identity and trafficking of enzymes necessary for the maturation and function of newly imported tRNAs. In particular, the aminoacyl-tRNA synthetases (aaRSs) that charge tRNAs are usually divided into distinct classes that specialize on either organellar (mitochondrial and plastid) or nuclear-encoded (cytosolic) tRNAs. Here, we investigate the evolution of aaRS subcellular localization in a plant lineage (Sileneae) that has experienced extensive and rapid mitochondrial tRNA loss. By analyzing full-length mRNA transcripts (PacBio Iso-Seq), we found predicted retargeting of many ancestrally cytosolic aaRSs to the mitochondrion and confirmed these results with colocalization microscopy assays. However, we also found cases where aaRS localization does not appear to change despite functional tRNA replacement, suggesting evolution of novel interactions and charging relationships. Therefore, the history of repeated tRNA replacement in Sileneae mitochondria reveals that differing constraints on tRNA/aaRS interactions may determine which of these alternative coevolutionary paths is used to maintain organellar translation in plant cells.

Delivery of AKT1 phospho-forms to human cells reveals differential substrate selectivity.

Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1-dependent activity in human cells, using a cell-penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho-variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl-transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT-tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT-tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT-pAKT1 variants induced glycogen synthase kinase (GSK)-3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT-pAKT1S473 or TAT-pAKT1T308,S473) were able to increase phospho-GSK-3ß levels. Although each TAT-pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT-pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho-forms in modulating downstream signaling and proliferation in human cells.