Distinctive physical properties of DNA shared by RNA polymerase II gene promoters and 5'-flanking regions of tRNA genes.

Numerous noncoding (nc)RNAs have been identified. Similar to the transcription of protein-coding (mRNA) genes, long noncoding (lnc)RNA genes and most of micro (mi)RNA genes are transcribed by RNA polymerase II (Pol II). In the transcription of mRNA genes, core promoters play an indispensable role; they support the assembly of the preinitiation complex (PIC). However, the structural and/or physical properties of the core promoters of lncRNA and miRNA genes remain largely unexplored, in contrast with those of mRNA genes. Using the core promoters of human genes, we analyzed the repertoire and population ratios of residing core promoter elements (CPEs) and calculated the following five DNA physical properties (DPPs): duplex DNA free energy, base stacking energy, protein-induced deformability, rigidity and stabilizing energy of Z-DNA. Here, we show that their CPE and DPP profiles are similar to those of mRNA gene promoters. Importantly, the core promoters of these three classes of genes have two highly distinctive sites in their DPP profiles around the TSS and position -27. Similar characteristics in DPPs are also found in the 5'-flanking regions of tRNA genes, indicating their common essential roles in transcription initiation over the kingdom of RNA polymerases.

tRNA-derived small RNAs in human cancers: roles, mechanisms, and clinical application.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are a new type of non-coding RNAs (ncRNAs) produced by the specific cleavage of precursor or mature tRNAs. tsRNAs are involved in various basic biological processes such as epigenetic, transcriptional, post-transcriptional, and translation regulation, thereby affecting the occurrence and development of various human diseases, including cancers. Recent studies have shown that tsRNAs play an important role in tumorigenesis by regulating biological behaviors such as malignant proliferation, invasion and metastasis, angiogenesis, immune response, tumor resistance, and tumor metabolism reprogramming. These may be new potential targets for tumor treatment. Furthermore, tsRNAs can exist abundantly and stably in various bodily fluids (e.g., blood, serum, and urine) in the form of free or encapsulated extracellular vesicles, thereby affecting intercellular communication in the tumor microenvironment (TME). Meanwhile, their abnormal expression is closely related to the clinicopathological features of tumor patients, such as tumor staging, lymph node metastasis, and poor prognosis of tumor patients; thus, tsRNAs can be served as a novel type of liquid biopsy biomarker. This review summarizes the discovery, production, and expression of tsRNAs and analyzes their molecular mechanisms in tumor development and potential applications in tumor therapy, which may provide new strategies for early diagnosis and targeted therapy of tumors.

Structural basis of translation inhibition by a valine tRNA-derived fragment.

Translational regulation by non-coding RNAs is a mechanism commonly used by cells to fine-tune gene expression. A fragment derived from an archaeal valine tRNA (Val-tRF) has been previously identified to bind the small subunit of the ribosome and inhibit translation in Haloferax volcanii Here, we present three cryo-electron microscopy structures of Val-tRF bound to the small subunit of Sulfolobus acidocaldarius ribosomes at resolutions between 4.02 and 4.53 Å. Within these complexes, Val-tRF was observed to bind to conserved RNA-interacting sites, including the ribosomal decoding center. The binding of Val-tRF destabilizes helices h24, h44, and h45 and the anti-Shine-Dalgarno sequence of 16S rRNA. The binding position of this molecule partially overlaps with the translation initiation factor aIF1A and occludes the mRNA P-site codon. Moreover, we found that the binding of Val-tRF is associated with steric hindrance of the H69 base of 23S rRNA in the large ribosome subunit, thereby preventing 70S assembly. Our data exemplify how tRNA-derived fragments bind to ribosomes and provide new insights into the mechanisms underlying translation inhibition by Val-tRFs.

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

Prophage terminase with tRNase activity sensitizes Salmonella enterica to oxidative stress.

Phage viruses shape the evolution and virulence of their bacterial hosts. The Salmonella enterica genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein, whose canonical function is to process phage DNA for packaging in the virus head, unexpectedly acts as a transfer ribonuclease (tRNase) under oxidative stress, cleaving the anticodon loop of tRNALeu. The ensuing RNA fragmentation compromises bacterial translation, intracellular survival, and recovery from oxidative stress in the vertebrate host. S. enterica adapts to this transfer RNA (tRNA) fragmentation by transcribing the RNA repair Rtc system. The counterintuitive translational arrest provided by tRNA cleavage may subvert prophage mobilization and give the host an opportunity for repair as a way of maintaining bacterial genome integrity and ultimately survival in animals.

Structure-based identification of salicylic acid derivatives as malarial threonyl tRNA-synthetase inhibitors.

Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Threonyl t-RNA synthetase (ThrRS) is one of the enzymes involved in this pathway, and it has been validated as an anti-malarial drug target. Here, we present 9 structurally diverse low micromolar Plasmodium falciparum ThrRS inhibitors that were identified using high-throughput virtual screening (HTVS) and were verified in a FRET enzymatic assay. Salicylic acid-based compound (LE = 0.34) was selected as a most perspective hit and was subjected to hit-to-lead optimisation. A total of 146 hit analogues were synthesised or obtained from commercial vendors and were tested. Structure-activity relationship study was supported by the crystal structure of the complex of a salicylic acid analogue with a close homologue of the plasmodium target, E. coli ThrRS (EcThrRS). Despite the availability of structural information, the hit identified via virtual screening remained one of the most potent PfThrRS inhibitors within this series. However, the compounds presented herein provide novel scaffolds for ThrRS inhibitors, which could serve as starting points for further medicinal chemistry projects targeting ThrRSs or structurally similar enzymes.

Clinical phenotype and genetic function analysis of a family with hypomyelinating leukodystrophy-7 caused by POLR3A mutation.

Hypomyelinating leukodystrophy (HLD) is a rare genetic heterogeneous disease that can affect myelin development in the central nervous system. This study aims to analyze the clinical phenotype and genetic function of a family with HLD-7 caused by POLR3A mutation. The proband (IV6) in this family mainly showed progressive cognitive decline, dentin dysplasia, and hypogonadotropic hypogonadism. Her three old brothers (IV1, IV2, and IV4) also had different degrees of ataxia, dystonia, or dysarthria besides the aforementioned manifestations. Their brain magnetic resonance imaging showed bilateral periventricular white matter atrophy, brain atrophy, and corpus callosum atrophy and thinning. The proband and her two living brothers (IV2 and IV4) were detected to carry a homozygous mutation of the POLR3A (NM_007055.4) gene c. 2300G > T (p.Cys767Phe), and her consanguineous married parents (III1 and III2) were p.Cys767Phe heterozygous carriers. In the constructed POLR3A wild-type and p.Cys767Phe mutant cells, it was seen that overexpression of wild-type POLR3A protein significantly enhanced Pol III transcription of 5S rRNA and tRNA Leu-CAA. However, although the mutant POLR3A protein overexpression was increased compared to the wild-type protein overexpression, it did not show the expected further enhancement of Pol III function. On the contrary, Pol III transcription function was frustrated (POLR3A, BC200, and tRNA Leu-CAA expression decreased), and MBP and 18S rRNA expressions were decreased. This study indicates that the POLR3A p.Cys767Phe variant caused increased expression of mutated POLR3A protein and abnormal expression of Pol III transcripts, and the mutant POLR3A protein function was abnormal.

Why tRNA acquisition could be relevant to bacteriophages?

In this opinion, we discuss the role of tRNAs in phage biology and their importance in DNA replication and phage-host interactions. Phages are a diverse group of obligate bacterial viruses that possess genomes with a wide range of sizes. Among them, we find phages with few genes that depend entirely on their host's translational machinery for replication. However, some phages carry genes for all replication steps and even contain genes for their own translational synthesis. In these cases, the integration of tRNA genes in their genomes is not completely understood, generating different theories about their presence and function during the replication cycle. Although different studies have attempted to elucidate their role, additional studies are needed to clarify the presence and significance of tRNA genes in phages. Moreover, we highlight the importance of tRNA genes in phages from both ecological and therapeutic perspectives.

Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Translation velocity determines the efficacy of engineered suppressor tRNAs on pathogenic nonsense mutations.

Nonsense mutations - the underlying cause of approximately 11% of all genetic diseases - prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon (PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.

Genetic Code Expansion in Pseudomonas putida KT2440.

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440.

It takes two to tRNAgo.

In this issue of Structure, Sievers et al.1 gain important insights into the human tRNA guanine transglycosylase QTRT1/2. The study presents a cryo-EM reconstruction of the inhibited human heterodimer in complex with human tRNAAsp, which represents the first snapshot of a eukaryotic TGT in complex with its full-length tRNA substrate.

Host-like RNA Elements Regulate Virus Translation.

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5' or 3' ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5' cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This "thrifty" virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3' end viral elements include 3'-cap-independent translation elements (3'-CITEs) and 3'-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity.

RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity.

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

2'-O-methylation (Nm) in RNA: progress, challenges, and future directions.

RNA 2'-O-methylation (Nm) is highly abundant in noncoding RNAs including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), and occurs in the 5' cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes. More recently, Nm has also been reported to occur at internal sites in mRNA. High-throughput methods have been developed for the transcriptome-wide detection of Nm. However, these methods have mostly been applied to abundant RNAs such as rRNA, and the validity of the internal mRNA Nm sites detected with these approaches remains controversial. Nonetheless, Nm in both coding and noncoding RNAs has been demonstrated to impact cellular processes, including translation and splicing. In addition, Nm modifications at the 5' cap and possibly at internal sites in mRNA serve to prevent the binding of nucleic acid sensors, thus preventing the activation of the innate immune response by self-mRNAs. Finally, Nm has been implicated in a variety of diseases including cancer, cardiovascular diseases, and neurologic syndromes. In this review, we discuss current challenges in determining the distribution, regulation, function, and disease relevance of Nm, as well as potential future directions for the field.

Chemical manipulation of m1A mediates its detection in human tRNA.

N 1-methyl adenosine (m1A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m1A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red-m1A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-GTT and 5.8S rRNA.

Bioorthogonal labeling and profiling of N6-isopentenyladenosine (i6A) modified RNA.

Chemical modifications in RNAs play crucial roles in diversifying their structures and regulating numerous biochemical processes. Since the 1990s, several hydrophobic prenyl-modifications have been discovered in various RNAs. Prenyl groups serve as precursors for terpenes and many other biological molecules. The processes of prenylation in different macromolecules have been extensively studied. We introduce here a novel chemical biology toolkit that not only labels i6A, a prenyl-modified RNA residue, by leveraging the unique reactivity of the prenyl group, but also provides a general strategy to incorporate fluorescence functionalities into RNAs for molecular tracking purposes. Our findings revealed that iodine-mediated cyclization reactions of the prenyl group occur rapidly, transforming i6A from a hydrogen-bond acceptor to a donor. Based on this reactivity, we developed an Iodine-Mediated Cyclization and Reverse Transcription (IMCRT) tRNA-seq method, which can profile all nine endogenous tRNAs containing i6A residues in Saccharomyces cerevisiae with single-base resolution. Furthermore, under stress conditions, we observed a decline in i6A levels in budding yeast, accompanied by significant decrease of mutation rate at A37 position. Thus, the IMCRT tRNA-seq method not only permits semi-quantification of i6A levels in tRNAs but also holds potential for transcriptome-wide detection and analysis of various RNA species containing i6A modifications.

Extended stop codon context predicts nonsense codon readthrough efficiency in human cells.

Protein synthesis terminates when a stop codon enters the ribosome's A-site. Although termination is efficient, stop codon readthrough can occur when a near-cognate tRNA outcompetes release factors during decoding. Seeking to understand readthrough regulation we used a machine learning approach to analyze readthrough efficiency data from published HEK293T ribosome profiling experiments and compared it to comparable yeast experiments. We obtained evidence for the conservation of identities of the stop codon, its context, and 3'-UTR length (when termination is compromised), but not the P-site codon, suggesting a P-site tRNA role in readthrough regulation. Models trained on data from cells treated with the readthrough-promoting drug, G418, accurately predicted readthrough of premature termination codons arising from CFTR nonsense alleles that cause cystic fibrosis. This predictive ability has the potential to aid development of nonsense suppression therapies by predicting a patient's likelihood of improvement in response to drugs given their nonsense mutation sequence context.

Beyond the Anticodon: tRNA Core Modifications and Their Impact on Structure, Translation and Stress Adaptation.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional chemical modifications. Approximately 100 different modifications have been identified in tRNAs, and each tRNA typically contains 5-15 modifications that are incorporated at specific sites along the tRNA sequence. These modifications may be classified into two groups according to their position in the three-dimensional tRNA structure, i.e., modifications in the tRNA core and modifications in the anticodon-loop (ACL) region. Since many modified nucleotides in the tRNA core are involved in the formation of tertiary interactions implicated in tRNA folding, these modifications are key to tRNA stability and resistance to RNA decay pathways. In comparison to the extensively studied ACL modifications, tRNA core modifications have generally received less attention, although they have been shown to play important roles beyond tRNA stability. Here, we review and place in perspective selected data on tRNA core modifications. We present their impact on tRNA structure and stability and report how these changes manifest themselves at the functional level in translation, fitness and stress adaptation.

Comparative Mitogenomics Analysis Revealed Evolutionary Divergence among Neopestalotiopsis Species Complex (Fungi: Xylariales).

The genus Neopestalotiopsis consists of obligate parasites that cause ring spot, scab, and leaf blight diseases in higher plant species. We assembled the three complete mitogenomes for the guava fruit ring spot pathogen, Neopestalotiopsis cubana. The mitogenomes are circular, with sizes of 38,666 bp, 33,846 bp, and 32,593 bp. The comparative analyses with Pestalotiopsis fici showed that N. cubana differs greatly from it in the length of the mitogenomes and the number of introns. Moreover, they showed significant differences in the gene content and tRNAs. The two genera showed little difference in gene skewness and codon preference for core protein-coding genes (PCGs). We compared gene sequencing in the mitogenomes of the order Xylariales and found large-scale gene rearrangement events, such as gene translocations and the duplication of tRNAs. N. cubana shows a unique evolutionary position in the phylum Ascomycota constructed in phylogenetic analyses. We also found a more concentrated distribution of evolutionary pressures on the PCGs of Neopestalotiopsis in the phylum Ascomycota and that they are under little selective pressure compared to other species and are subjected to purifying selection. This study explores the evolutionary dynamics of the mitogenomes of Neopestalotiopsis and provides important support for genetic and taxonomic studies.