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Type 2 diabetes mellitus (T2D) affects millions of people worldwide and is one of the leading causes of morbidity and mortality. The skeletal muscle (SKM) is the most important tissue involved in maintaining glucose homeostasis and substrate oxidation, and it undergoes insulin resistance in T2D. In this study, we identify the existence of alterations in the expression of mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in skeletal muscle from two different forms of T2D: early-onset type 2 diabetes (YT2) (onset of the disease before 30 years of age) and the classical form of the disease (OT2). GSEA analysis from microarray studies revealed the repression of mitochondrial mt-aaRSs independently of age, which was validated by real-time PCR assays. In agreement with this, a reduced expression of several encoding mt-aaRSs was also detected in skeletal muscle from diabetic (db/db) mice but not in obese ob/ob mice. In addition, the expression of the mt-aaRSs proteins most relevant in the synthesis of mitochondrial proteins, threonyl-tRNA, and leucyl-tRNA synthetases (LARS2 and TARS2) were also repressed in muscle from db/db mice. It is likely that these alterations participate in the reduced expression of proteins synthesized in the mitochondria detected in db/db mice. Because it is known that, nitrosative stress inhibits aminoacylation of TARS2 and LARS2 activities, we noticed an increased protein expression of iNOS in isolated muscle mitochondria in diabetic mice.Our results indicate a reduced expression of mitochondrial mt-aaRSs in skeletal muscle from T2D patients, which may participate in the reduced expression of proteins synthesized in mitochondria. This may be due to an enhanced NO production secondary to enhanced iNOS expression in muscle under diabetic conditions.HighlightsMt-aaRSs are downregulated in the skeletal muscle of type 2 diabetic patients and diabetic mice.The downregulation of mt-aaRSs in the skeletal muscle of diabetic mice is affecting the synthesis of ND2 which is amitochondriallyencodedsubunitof complex I.Mitochondrial iNOS could be a target for reduced expression of mt-aaRSs in the skeletal muscle of diabetic mice.

The genetic code is the manual that cells use to incorporate amino acids into proteins. It is possible to artificially expand this manual through cellular, molecular, and chemical manipulations to improve protein functionality. Strategies for in vivo genetic code expansion are under the same functional constraints as natural protein synthesis. Here, we review the approaches used to incorporate noncanonical amino acids (ncAAs) into designer proteins through the manipulation of the translation machinery and draw parallels between these methods and natural adaptations that improve translation in extant organisms. Following this logic, we propose new nature-inspired tactics to improve genetic code expansion (GCE) in synthetic organisms.

Summary High-throughput sequencing of transfer RNAs (tRNA-Seq) is a powerful approach to characterize the cellular tRNA pool. Currently, however, analyzing tRNA-Seq datasets requires strong bioinformatics and programming skills. tRNAstudio facilitates the analysis of tRNA-Seq datasets and extracts information on tRNA gene expression, post-transcriptional tRNA modification levels, and tRNA processing steps. Users need only running a few simple bash commands to activate a graphical user interface that allows the easy processing of tRNA-Seq datasets in local mode. Output files include extensive graphical representations and associated numerical tables, and an interactive html summary report to help interpret the data. We have validated tRNAstudio using datasets generated by different experimental methods and derived from human cell lines and tissues that present distinct patterns of tRNA expression, modification and processing. Availability and implementation Freely available at https://github.com/GeneTranslationLab-IRB/tRNAstudio under an open-source GNU GPL v3.0 license. Supplementary information Supplementary data are available at Bioinformatics online.

The human genome encodes hundreds of transfer RNA (tRNA) genes but their individual contribution to the tRNA pool is not fully understood. Deep sequencing of tRNA transcripts (tRNA-Seq) can estimate tRNA abundance at single gene resolution, but tRNA structures and posttranscriptional modifications impair these analyses. Here we present a bioinformatics strategy to investigate differential tRNA gene expression and use it to compare tRNA-Seq datasets from cultured human cells and human brain. We find that sequencing caveats affect quantitation of only a subset of human tRNA genes. Unexpectedly, we detect several cases where the differences in tRNA expression among samples do not involve variations at the level of isoacceptor tRNA sets (tRNAs charged with the same amino acid but using different anticodons), but rather among tRNA genes within the same isodecoder set (tRNAs having the same anticodon sequence). Because isodecoder tRNAs are functionally equal in terms of genetic translation, their differential expression may be related to noncanonical tRNA functions. We show that several instances of differential tRNA gene expression result in changes in the abundance of tRNA-derived fragments (tRFs) but not of mature tRNAs. Examples of differentially expressed tRFs include PIWI-associated RNAs, tRFs present in tissue samples but not in cells cultured in vitro, and somatic tissue-specific tRFs. Our data support that differential expression of tRNA genes regulate noncanonical tRNA functions performed by tRFs.

The modification of adenosine to inosine at the wobble position (I34) of tRNA anticodons is an abundant and essential feature of eukaryotic tRNAs. The expansion of inosine-containing tRNAs in eukaryotes followed the transformation of the homodimeric bacterial enzyme TadA, which generates I34 in tRNAArg and tRNALeu, into the heterodimeric eukaryotic enzyme ADAT, which modifies up to eight different tRNAs. The emergence of ADAT and its larger set of substrates, strongly influenced the tRNA composition and codon usage of eukaryotic genomes. However, the selective advantages that drove the expansion of I34-tRNAs remain unknown. Here we investigate the functional relevance of I34-tRNAs in human cells and show that a full complement of these tRNAs is necessary for the translation of low-complexity protein domains enriched in amino acids cognate for I34-tRNAs. The coding sequences for these domains require codons translated by I34-tRNAs, in detriment of synonymous codons that use other tRNAs. I34-tRNA-dependent low-complexity proteins are enriched in functional categories related to cell adhesion, and depletion in I34-tRNAs leads to cellular phenotypes consistent with these roles. We show that the distribution of these low-complexity proteins mirrors the distribution of I34-tRNAs in the phylogenetic tree. Spanish Ministry of Economy and Competitiveness [PID2019-108037RB-100 to L.R.d.P., PGC2018-099921 to T.G., PGC2018-098152-A-100 to E.M.N]; Australian Research Council [DP180103571 to E.M.N]; European Union's Horizon 2020 research and innovation programme [ERC-2016–724173 to T.G.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness. Peer Reviewed "Article signat per 14 autors/es: Adrian Gabriel Torres, Marta Rodríguez-Escribà, Marina Marcet-Houben, Helaine Graziele Santos Vieira, Noelia Camacho, Helena Catena, Marina Murillo Recio, Àlbert Rafels-Ybern, Oscar Reina, Francisco Miguel Torres, Ana Pardo-Saganta, Toni Gabaldón, Eva Maria Novoa, Lluís Ribas de Pouplana"

International audience; BRCA1 inactivation is a hallmark of familial breast cancer, often associated with aggressive triple negative breast cancers. BRCA1 is a tumor suppressor with known functions in DNA repair, transcription regulation, cell cycle control, and apoptosis. In the present study, we demonstrate that BRCA1 is also a translational regulator. We previously showed that BRCA1 was implicated in translation regulation. Here, we asked whether translational control could be a novel function of BRCA1 that contributes to its tumor suppressive activity. A combination of RNA-binding protein immunoprecipitation, microarray analysis, and polysome profiling, was used to identify the mRNAs that were specifically deregulated under BRCA1 deficiency. Western blot analysis allowed us to confirm at the protein level the deregulated translation of a subset of mRNAs. A unique and dedicated cohort of patients with documented germ-line BRCA1 pathogenic variant statues was set up, and tissue microarrays with the biopsies of these patients were constructed and analyzed by immunohistochemistry for their content in each candidate protein. Here, we show that BRCA1 translationally regulates a subset of mRNAs with which it associates. These mRNAs code for proteins involved in major programs in cancer. Accordingly, the level of these key proteins is correlated with BRCA1 status in breast cancer cell lines and in patient breast tumors. ADAT2, one of these key proteins, is proposed as a predictive biomarker of efficacy of treatments recently recommended to patients with BRCA1 deficiency. This study proposes that translational control may represent a novel molecular mechanism with potential clinical impact through which BRCA1 is a tumor suppressor.

Inosine at the "wobble" position (I34) is one of the few essential posttranscriptional modifications in tRNAs (tRNAs). It results from the deamination of adenosine and occurs in bacteria on tRNAArgACG and in eukarya on six or seven additional tRNA substrates. Because inosine is structurally a guanosine analogue, reverse transcriptases recognize it as a guanosine. Most methods used to examine the presence of inosine rely on this phenomenon and detect the modified base as a change in the DNA sequence that results from the reverse transcription reaction. These methods, however, cannot always be applied to tRNAs because reverse transcription can be compromised by the presence of other posttranscriptional modifications. Here we present SL-ID (splinted ligation-based inosine detection), a reverse transcription-free method for detecting inosine based on an I34-dependent specific cleavage of tRNAs by endonuclease V, followed by a splinted ligation and polyacrylamide gel electrophoresis analysis. We show that the method can detect I34 on different tRNA substrates and can be applied to total RNA derived from different species, cell types, and tissues. Here we apply the method to solve previous controversies regarding the modification status of mammalian tRNAArgACG.

The modification of adenosine to inosine at position 34 of tRNA anticodons has a profound impact upon codon-anticodon recognition. In bacteria, I34 is thought to exist only in tRNAArg, while in eukaryotes the modification is present in eight different tRNAs. In eukaryotes, the widespread use of I34 strongly influenced the evolution of genomes in terms of tRNA gene abundance and codon usage. In humans, codon usage indicates that I34 modified tRNAs are preferred for the translation of highly repetitive coding sequences, suggesting that I34 is an important modification for the synthesis of proteins of highly skewed amino acid composition. Here we extend the analysis of distribution of codons that are recognized by I34 containing tRNAs to all phyla known to use this modification. We find that the preference for codons recognized by such tRNAs in genes with highly biased codon compositions is universal among eukaryotes, and we report that, unexpectedly, some bacterial phyla show a similar preference. We demonstrate that the genomes of these bacterial species contain previously undescribed tRNA genes that are potential substrates for deamination at position 34.

The nucleoside inosine plays an important role in purine biosynthesis, gene translation, and modulation of the fate of RNAs. The editing of adenosine to inosine is a widespread post-transcriptional modification in transfer RNAs (tRNAs) and messenger RNAs (mRNAs). At the wobble position of tRNA anticodons, inosine profoundly modifies codon recognition, while in mRNA, inosines can modify the sequence of the translated polypeptide or modulate the stability, localization, and splicing of transcripts. Inosine is also found in non-coding and exogenous RNAs, where it plays key structural and functional roles. In addition, molecular inosine is an important secondary metabolite in purine metabolism that also acts as a molecular messenger in cell signaling pathways. Here, we review the functional roles of inosine in biology and their connections to human health.

International audience; The rate at which ribosomes translate mRNAs regulates protein expression by controlling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage and phosphorylation of eukaryotic elongation factor 2 (eEF2). Current methods to measure translation elongation lack single-cell resolution, require expression of multiple transgenes and have never been successfully applied ex vivo. Here, we show, by using a combination of puromycilation detection and flow cytometry (a method we call 'SunRiSE'), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues. This method allows for the simultaneous monitoring of multiple parameters, such as mTOR or S6K1/2 signaling activity, the cell cycle stage and phosphorylation of translation factors in single cells, without elaborated, costly and lengthy purification procedures. We took advantage of SunRiSE to demonstrate that, in mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase (eEF2K) mostly affects translation engagement, but has a surprisingly small effect on elongation, except after proteotoxic stress induction. This article has an associated First Person interview with the first author of the paper.

Transfer RNAs (tRNAs) are essential components of the protein translation machinery. In order to become fully active, they need to be heavily modified post-transcriptionally. Such modifications affect the structure, stability and functionality of tRNAs; however, their exact roles at the molecular level remain largely elusive. Here we focus on the biological functions of tRNA modifications associated to human diseases and how such information can be used for biomedical applications. We put an emphasis on mitochondrial-linked dysfunctions, metabolic disorders, neurological defects and cancer. We also present methods and approaches currently used in the clinic to detect and monitor different human pathologies involving tRNA modifications or tRNA modification enzymes, and, additionally, we propose novel tRNA modification-based strategies that could be used for diagnosis, prognosis or treatment of human diseases.

The size of the genetic code is limited by the ability of transfer RNAs to acquire new identities., Understanding the principles that led to the current complexity of the genetic code is a central question in evolution. Expansion of the genetic code required the selection of new transfer RNAs (tRNAs) with specific recognition signals that allowed them to be matured, modified, aminoacylated, and processed by the ribosome without compromising the fidelity or efficiency of protein synthesis. We show that saturation of recognition signals blocks the emergence of new tRNA identities and that the rate of nucleotide substitutions in tRNAs is higher in species with fewer tRNA genes. We propose that the growth of the genetic code stalled because a limit was reached in the number of identity elements that can be effectively used in the tRNA structure.

Efficient cell delivery of antisense oligonucleotides (ONs) is a key issue for their potential therapeutic use. It has been shown recently that some ONs can be delivered into cells without the use of transfection agents (gymnosis), but this generally requires cell incubation over several days and high amounts of ONs (micromolar concentrations). Here we have targeted microRNA 122 (miR-122), a small non-coding RNA involved in regulation of lipid metabolism and in the replication of hepatitis C virus, with ONs of different chemistries (anti-miRs) by gymnotic delivery in cell culture. Using a sensitive dual-luciferase reporter assay, anti-miRs were screened for their ability to enter liver cells gymnotically and inhibit miR-122 activity. Efficient miR-122 inhibition was obtained with cationic PNAs and 2'-O-methyl (OMe) and Locked Nucleic Acids (LNA)/OMe mixmers containing either phosphodiester (PO) or phosphorothioate (PS) linkages at sub-micromolar concentrations when incubated with cells for just 4 hours. Furthermore, PNA and PS-containing anti-miRs were able to sustain miR-122 inhibitory effects for at least 4 days. LNA/OMe PS anti-miRs were the most potent anti-miR chemistry tested in this study, an ON chemistry that has been little exploited so far as anti-miR agents towards therapeutics.

Transfer RNAs (tRNAs) are key adaptor molecules of the genetic code that are heavily modified post-transcriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential widespread tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalyzed by the heterodimeric enzyme adenosine deaminase acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing (RNAseq), we show that this modification is incorporated to human tRNAs at the precursor tRNA level and during maturation. We also functionally validated the human genes encoding for hetADAT and show that the subunits of this enzyme co-localize in nucleus in an ADAT2-dependent manner. Finally, by knocking down HsADAT2, we demonstrate that variations in the cellular levels of hetADAT will result in changes in the levels of I34 modification in all its potential substrates. Altogether, we present RNAseq as a powerful tool to study post-transcriptional tRNA modifications at the precursor tRNA level and give the first insights on the biology of I34 tRNA modification in metazoans.

The frozen accident theory of the Genetic Code was a proposal by Francis Crick that attempted to explain the universal nature of the Genetic Code and the fact that it only contains information for twenty amino acids. Fifty years later, it is clear that variations to the universal Genetic Code exist in nature and that translation is not limited to twenty amino acids. However, given the astonishing diversity of life on earth, and the extended evolutionary time that has taken place since the emergence of the extant Genetic Code, the idea that the translation apparatus is for the most part immobile remains true. Here, we will offer a potential explanation to the reason why the code has remained mostly stable for over three billion years, and discuss some of the mechanisms that allow species to overcome the intrinsic functional limitations of the protein synthesis machinery.

Inosine on transfer RNAs (tRNAs) are post-transcriptionally formed by a deamination mechanism of adenosines at positions 34, 37 and 57 of certain tRNAs. Despite its ubiquitous nature, the biological role of inosine in tRNAs remains poorly understood. Recent developments in the study of nucleotide modifications are beginning to indicate that the dynamics of such modifications are used in the control of specific genetic programs. Likewise, the essentiality of inosine-modified tRNAs in genome evolution and animal biology is becoming apparent. Here we review our current understanding on the role of inosine in tRNAs, the enzymes that catalyze the modification and the evolutionary link between such enzymes and other deaminases.

Faithful translation of genomes into proteomes depends, mainly, on the activity of transfer RNA (tRNA) as universal adaptor, as insightfully predicted by Crick. The central role of this relatively simple oligonucleotide depends upon a very large number of intermolecular interactions, some of which require that tRNAs maintain a constant general structure, while others depend on specific features that discriminate any given tRNA from the rest. Posttranscriptional modifications that increase the chemical diversity contained in the nucleotides of tRNAs can serve both purposes. Chemical modifications of tRNAs, thus, come in two general flavors: those that help to maintain the general shape of the molecule, and those that improve its interactions with one or more of its many molecular partners. Although the function of most of the chemical modifications known to occur in tRNAs remain unknown, up-to-date knowledge allows us to analyze the majority of them in some model organisms, including Saccharomyces cerevisiae. Here we will review our current understanding on the function of tRNA modifications, generally dividing them into two families: those that are likely to influence the structure of tRNA, and those that may play a role in the codon-anticodon interaction at the decoding center of the ribosome.

Two vitellogenin genes (Vg1 and Vg2) were identified in the Chagas' disease vector Triatoma infestans. The putative coding sequence corresponding to Vg2 was found to be 5553 bp long, encoding 1851 amino acids in a single open reading frame. The comparative analysis of the deduced amino acid sequences from Vg1 and Vg2 cDNA fragments of T. infestans revealed 58.94% of identity with 76.43% of homology. The phylogenetic tree based on the complete Vg amino acid sequences of hemimetabolous insects unambiguously supported two clusters, one consisting of Vg sequences from dictyopteran and the other containing Vg sequences of hemipteran. The Vg1 and Vg2 mRNAs were detected in fat bodies and ovaries of adult females with the highest levels of both Vg transcripts in the first tissue. Quantitative PCR showed low expression of Vg2 in head and muscle of adult females, while the Vg1 transcript was not present in these organs. Neither Vg1 nor Vg2 was expressed in fifth instar nymph fat bodies or in adult male fat bodies, heads, and muscles. Fil: Blariza, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Soria, Néstor Walter. Laboratorio de Análisis Clínicos Especializados; Argentina Fil: Torres, Adrian G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Grosso, Carla Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Garcia, Beatriz Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina

Transfer RNAs (tRNAs) are key for efficient and accurate protein translation. To be fully active, tRNAs need to be heavily modified post-transcriptionally. Growing evidence indicates that tRNA modifications and the enzymes catalyzing such modifications may play important roles in complex human pathologies. Here, we have compiled current knowledge that directly link tRNA modifications to human diseases such as cancer, type 2 diabetes (T2D), neurological disorders, and mitochondrial-linked disorders. The molecular mechanisms behind these connections remain, for the most part, unknown. As we progress towards the understanding of the roles played by hypomodified tRNAs in human disease, novel areas of therapeutic intervention may be discovered.

Chimeric 2'-O-methyl oligoribonucleotides (2'-OMe ORNs) containing internucleotide linkages which were modified with phosphonoacetate (PACE) or thiophosphonoacetate (thioPACE) were prepared by solid-phase synthesis. The modified 2'-OMe ORNs contained a central phosphate or phosphorothioate sequence with up to 4 PACE or thioPACE modifications, respectively, at either end of the ORN in a "gapmer" motif. Both PACE and thioPACE 2'-OMe ORNs formed stable duplexes with complementary RNA. The majority of these duplexes had higher thermal melting temperatures than an unmodified RNA:RNA duplex. The modified 2'-OMe ORNs were effective passenger strands with complementary, unmodified siRNAs, for inducing siRNA activity in a dual luciferase assay in the presence of a lipid transfecting agent. As single strands, thioPACE 2'-OMe ORNs were efficiently taken up by HeLa cells in the absence of a lipid transfecting agent. Furthermore, thioPACE modifications greatly improved the potency of a 2'-OMe phosphorothioate ORN as an inhibitor of microRNA-122 in Huh7 cells, without lipid transfection.

Efficient cellular delivery is one of the key issues that has hampered the therapeutic development of novel synthetic biomolecules such as oligonucleotides, peptides and nanoparticles. The highly specialized cellular plasma membrane specifically internalizes compounds through tightly regulated mechanisms. It is possible to exploit these natural mechanisms of cellular uptake with rationally designed reagents. Here, we discuss how thiol groups (-SH) naturally present on the cell surface (exofacial thiols) can be used to enhance cellular association and internalization of various materials bearing thiol-reactive groups in their structure. We propose that such thiol modifications should be considered in future design of synthetic biomolecules for optimized cellular delivery.

Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.

MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2′-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs.

MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use.

Variation in the mtDNA 16S ribosomal RNA gene in populations of Triatoma infestans (Klug) was surveyed. DNA sequence comparisons yielded 18 haplotypes among 130 individuals from 16 localities that represent a large proportion of the range of T. infestans in Argentina. The most common genotype in all populations was found in 76.9% of individuals and two other haplotypes were shared among different populations. The remaining 15 haplotypes were present exclusively in one of the populations, suggesting currently low levels of genetic exchange. Analysis of mtDNA 16S sequences uncovered substantial genetic variation among T. infestans populations. Haplotype and nucleotide diversities varied among populations, from 0% to 0.84% and 0% to 0.29%, respectively. It appears that this locus has a low mutation rate. Uncorrected pairwise differences of T. infestans haplotypes ranged from 0% to 1.2%. The molecular phylogeny supported the monophyly of T. infestans haplotypes and clustered two different pairs of haplotypes with a moderate degree of bootstrap support (approximately 60%). Mitochondrial DNA phylogeographic differentiation was not evident, suggesting a recent rapid spread of the species. Analysis of molecular variance showed hierarchical structure in the data. Considerably less variation was found among T. infestans populations from the northwest and northeast regions than among those belonging to the central area. Such a lack of variation may be indicative of one or more past population bottlenecks.

Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive, and laborious. Restriction fragment length polymorphism (RFLP) analyses study the patterns of DNA cleavage after restriction enzyme treatment and have been used for the qualitative detection of modified bases on mRNAs. It is known that some ptRMs induce specific and reproducible base "mutations" when tRNAs are reverse transcribed. For example, inosine, which derives from the deamination of adenosine, is detected as a guanosine when an inosine-containing tRNA is reverse transcribed, amplified via polymerase chain reaction (PCR), and sequenced. ptRM-dependent base changes on reverse transcription PCR amplicons generated as a consequence of the reverse transcription reaction might create or abolish endonuclease restriction sites. The suitability of RFLP for the detection and/or quantification of ptRMs has not been studied thus far. Here we show that different ptRMs can be detected at specific sites of different tRNA types by RFLP. For the examples studied, we show that this approach can reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications in gene expression.

A mutation in the WDR4 gene, coding for a tRNA-modifying enzyme, leads to reduced levels of guanosine methylation in tRNA in patients with primordial dwarfism. See related Research article: http://www.genomebiology.com/2015/16/1/210.