The lifecycle of retroviruses and retrotransposons includes a reverse transcription step, wherein dsDNA is synthesized from genomic RNA for subsequent insertion into the host genome. Retroviruses and ...retrotransposons commonly appropriate major components of the host cell translational machinery, including cellular tRNAs, which are exploited as reverse transcription primers. Nonpriming functions of tRNAs have also been proposed, such as in HIV-1 virion assembly, and tRNA-derived fragments may also be involved in retrovirus and retrotransposon replication. Moreover, host cellular proteins regulate retroviral replication by binding to tRNAs and thereby affecting various steps in the viral lifecycle. For example, in some cases, tRNA primer selection is facilitated by cognate aminoacyl-tRNA synthetases (ARSs), which bind tRNAs and ligate them to their corresponding amino acids, but also have many known nontranslational functions. Multi-omic studies have revealed that ARSs interact with both viral proteins and RNAs and potentially regulate retroviral replication. Here, we review the currently known roles of tRNAs and their derivatives in retroviral and retrotransposon replication and shed light on the roles of tRNA-binding proteins such as ARSs in this process.
Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to cognate tRNAs for use in protein synthesis. This historical function of ARSs and tRNAs is fairly well understood. ...However, ARSs and tRNAs also perform noncanonical functions that are continuing to be unveiled at a rapid pace. The expanded functions of these essential molecules of life range from roles in retroviral replication to stimulation of mammalian target of rapamycin (mTOR) activity; DNA repair, splicing, and transcriptional and translational regulation; and other aspects of cellular homeostasis. Furthermore, mutations in tRNAs and synthetases are known to drive human maladies, such as the neurodegenerative disorder Charcot-Marie-Tooth disease along with other central nervous system dysfunctions and cancer. This series of reviews focuses on the diseases that result from natural variations in human cytoplasmic tRNAs, as well as from mutations in mitochondrial tRNAs and ARSs. Ultimately, the exciting work in this rapidly emerging area may lead to new therapies for microbial and parasitic infections, cancer, and neurodegenerative diseases.
Abstract The RNA World hypothesis posits that RNA can represent a primitive life form by reproducing itself and demonstrating catalytic activity. However, this hypothesis is incapable of addressing ...several major origin‐of‐life (OoL) questions. A recently described paradox‐free alternative OoL hypothesis, the Quadruplex (G4) World, is based on the ability of poly(dG) to fold into a stable architecture with an unambiguous folding pattern using G‐tetrads as building elements. Because of the folding pattern of three G‐tetrads and single‐G loops, dG 15 is programmable and has the capability to encode biological information. Here, we address two open questions of the G4 World hypothesis: (1) Does RNA follow the same folding pattern as DNA? (2) How do stable quadruplexes evolve into the present‐day system of information transfer, which is based on Watson‐Crick base pair complementarity? To address these questions, we systematically studied the thermodynamic and optical properties of both DNA and RNA G15‐ and G3T (GGGTGGGTGGGTGGG)‐derived sequences. Our study revealed that similar to DNA sequences, RNAs adopt quadruplexes with only three G‐tetrads. Thus, both poly(dG) and poly(rG) possess inherent ability to fold into 3D quadruplex architecture with strictly defined folding pattern. The study also revealed that despite high stability of both DNA and RNA quadruplexes, they are vulnerable to single‐nucleotide substitutions, which drop the thermal stability by ~40°C and can facilitate introduction of the complementarity principle into the G4 World.
Abstract
Aminoacyl‐tRNA synthetases (aaRSs) maintain translational fidelity by ensuring the formation of correct aminoacyl‐tRNA pairs. Numerous point mutations in human aaRSs have been linked to ...disease phenotypes. Structural studies of aaRSs from human pathogens encoding unique domains support these enzymes as potential candidates for therapeutics. Studies have shown that the identity of tRNA pools in cells changes between different cell types and under stress conditions. While traditional radioactive aminoacylation analyses can determine the effect of disease‐causing mutations on aaRS function, these assays are not amenable to drug discovery campaigns and do not take into account the variability of the intracellular tRNA pools. Here, we review modern techniques to characterize aaRS activity
in vitro
and in cells. The cell‐based approaches analyse the aminoacyl‐tRNA pool to observe trends in aaRS activity and fidelity. Taken together, these approaches allow high‐throughput drug screening of aaRS inhibitors and systems‐level analyses of the dynamic tRNA population under a variety of conditions and disease states.
Aminoacyl‐tRNA synthetases are enzymes that ensure accurate protein synthesis. Variants of the dual‐functional cytoplasmic human glutamyl‐prolyl‐tRNA synthetase, EPRS1, have been associated with ...leukodystrophy, diabetes and bone disease. Here, we report compound heterozygous variants in EPRS1 in a 4‐year‐old female patient presenting with psychomotor developmental delay, seizures and deafness. Functional studies of these two missense mutations support major defects in enzymatic function in vitro and contributed to confirmation of the diagnosis.
We report compound heterozygous variants in a bifunctional aminoacyl‐tRNA synthetase, EPRS1, in a 4‐year‐old female patient presenting with psychomotor developmental delay, seizures and deafness. Functional studies of these two missense mutations support major defects in enzymatic function in vitro and contributed to confirmation of the diagnosis.
The Arabidopsis thaliana tandem zinc finger 1 (AtTZF1) protein is characterized by two tandem‐arrayed CCCH‐type zinc fingers. We have previously found that AtTZF1 affects hormone‐mediated growth, ...stress and gene expression responses. While much has been learned at the genetic and physiological level, the molecular mechanisms underlying the effects of AtTZF1 on gene expression remain obscure. A human TZF protein, hTTP, is known to bind and trigger the degradation of mRNAs containing AU‐rich elements (AREs) at the 3′ untranslated regions. However, while the TZF motif of hTTP is characterized by CX₈CX₅CX₃H‐X₁₈‐CX₈CX₅CX₃H, AtTZF1 contains an atypical motif of CX₇CX₅CX₃H‐X₁₆‐CX₅CX₄CX₃H. Moreover, the TZF motif of AtTZF1 is preceded by an arginine‐rich (RR) region that is unique to plants. Using fluorescence anisotropy and electrophoretic mobility shift binding assays, we have demonstrated that AtTZF1 binds to RNA molecules with specificity and the interaction is dependent on the presence of zinc. Compared with hTTP, in which TZF is solely responsible for RNA binding, both TZF and RR regions of AtTZF1 are required to achieve high‐affinity RNA binding. Moreover, zinc finger integrity is vital for RNA binding. Using a plant protoplast transient expression analysis we have further revealed that AtTZF1 can trigger the decay of ARE‐containing mRNAs in vivo. Taken together, our results support the notion that AtTZF1 is involved in RNA turnover.
Nucleic acid quadruplexes are proposed to play a role in the regulation of gene expression, are often present in aptamers selected for specific binding functions and have potential applications in ...medicine and biotechnology. Therefore, understanding their structure and thermodynamic properties and designing highly stable quadruplexes is desirable for a variety of applications. Here, we evaluate DNA→RNA substitutions in the context of a monomolecular, antiparallel quadruplex, the thrombin‐binding aptamer (TBA, GGTTGGTGTGGTTGG) in the presence of either K+ or Sr2+. TBA predominantly folds into a chair‐type configuration containing two G‐tetrads, with G residues in both syn and anti conformation. All chimeras with DNA→RNA substitutions (G→g) at G residues requiring the syn conformation demonstrated strong destabilization. In contrast, G→g substitutions at Gs with anti conformation increased stability without affecting the monomolecular chair‐type topology. None of the DNA→RNA substitutions in loop positions affected the quadruplex topology; however, these substitutions varied widely in their stabilizing or destabilizing effects in an unpredictable manner. This analysis allowed us to design a chimeric DNA/RNA TBA construct that demonstrated substantially improved stability relative to the all‐DNA construct. These results have implications for a variety of quadruplex‐based applications including for the design of dynamic nanomachines.
DNA→RNA substitutions in the context of a monomolecular, antiparallel quadruplex, the thrombin binding aptamer (TBA) have been investigated in the presence of either K+ or Sr2+. Analysis of the stabilizing and destabilizing influences allowed to design a chimeric DNA/RNA TBA construct that demonstrated substantially improved stability relative to the all‐DNA construct.
Aminoacyl-tRNA synthetases (AARSs) play a pivotal role in protein synthesis and cell viability. These 22 "housekeeping" enzymes (1 for each standard amino acid plus pyrrolysine and o-phosphoserine) ...are specifically involved in recognizing and aminoacylating their cognate tRNAs in the cellular pool with the correct amino acid prior to delivery of the charged tRNA to the protein synthesis machinery. Besides serving this canonical function, higher eukaryotic AARSs, some of which are organized in the cytoplasm as a multisynthetase complex of nine enzymes plus additional cellular factors, have also been implicated in a variety of non-canonical roles. AARSs are involved in the regulation of transcription, translation, and various signaling pathways, thereby ensuring cell survival. Based in part on their versatility, AARSs have been recruited by viruses to perform essential functions. For example, host synthetases are packaged into some retroviruses and are required for their replication. Other viruses mimic tRNA-like structures in their genomes, and these motifs are aminoacylated by the host synthetase as part of the viral replication cycle. More recently, it has been shown that certain large DNA viruses infecting animals and other diverse unicellular eukaryotes encode tRNAs, AARSs, and additional components of the protein-synthesis machinery. This chapter will review our current understanding of the role of host AARSs and tRNA-like structures in viruses and discuss their potential as anti-viral drug targets. The identification and development of compounds that target bacterial AARSs, thereby serving as novel antibiotics, will also be discussed. Particular attention will be given to recent work on a number of tRNA-dependent AARS inhibitors and to advances in a new class of natural "pro-drug" antibiotics called Trojan Horse inhibitors. Finally, we will explore how bacteria that naturally produce AARS-targeting antibiotics must protect themselves against cell suicide using naturally antibiotic resistant AARSs, and how horizontal gene transfer of these AARS genes to pathogens may threaten the future use of this class of antibiotics.
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•Features of tRNA contributing to inhibition of HIV-1 Gag membrane binding are unclear.•Membrane binding of purified MA is strongly inhibited by tRNALys3 but not tRNAPro.•The D arm ...but not anticodon arm contributes to strong inhibition by tRNALys3.•The D arm sequence and its context are major determinants for the inhibitory effect.
Plasma-membrane-specific localization of Gag, an essential step in HIV-1 particle assembly, is regulated by the interaction of the Gag MA domain with PI(4,5)P2 and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P2-independent membrane binding to varying degrees in vitro; however, the structural determinants for this difference remain unknown. Here we demonstrate that membrane binding of full-length Gag synthesized in vitro using reticulocyte lysates is inhibited when RNAs that contain the anticodon arm of tRNAPro, but not that of tRNALys3, are added exogenously. In contrast, in the context of a liposome binding assay in which the effects of tRNAs on purified MA were tested, full-length tRNALys3 showed greater inhibition of MA membrane binding than full-length tRNAPro. While transplantation of the D loop sequence of tRNALys3 into tRNAPro resulted in a modest increase in the inhibitory effect relative to WT tRNAPro, replacing the entire D arm sequence with that of tRNALys3 was necessary to confer the full inhibitory effects upon tRNAPro. Together, these results demonstrate that the D arm of tRNALys3 is a major determinant of strong inhibition of MA membrane binding and that this inhibitory effect requires not only the D loop, which was recently reported to contact the MA highly basic region, but the loop sequence in the context of the D arm structure.
Transfer RNAs: A treasure trove that keeps on giving Gopalan, Venkat; Musier-Forsyth, Karin
Journal of biological chemistry/The Journal of biological chemistry,
10/2023, Volume:
299, Issue:
10
Journal Article
Peer reviewed
Open access
Transfer RNAs are the adaptors in protein synthesis that provide the key link between the nucleic acid-based genetic blueprint and proteins. While the central role of tRNAs in protein synthesis has ...been known for over 60 years, recent discoveries of their many non-canonical functions and therapeutic potential have heightened interest in tRNAs. In this thematic series, we highlight some of the developments presented at the recent biennial “International tRNA Workshop”. The topics chosen reflect advances that were enabled by the latest technological breakthroughs in structure determination and small RNA sequencing and emphasize the prospects and challenges of tRNA-based medicines to treat human diseases.
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