Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus.

The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analysed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA. However, orthogonal validation by Illumina RNA-seq analyses did not identify any inosine modification along the CHIKV RNA genome. Moreover, CHIKV infection did not alter the expression of ADAR1 isoforms, the enzymes that catalyse the adenosine to inosine conversion. Together, this study highlights the importance of a multidisciplinary approach to assess the presence of RNA modifications in viral RNA genomes.

N6-methyladenosine modification is not a general trait of viral RNA genomes.

Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m6A in CHIKV and DENV RNAs. For this, we combine m6A-Seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we find no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m6A machinery's localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

Hepatocyte-specific O-GlcNAc transferase downregulation ameliorates nonalcoholic steatohepatitis by improving mitochondrial function.

OBJECTIVE: O-GlcNAcylation is a post-translational modification that directly couples the processes of nutrient sensing, metabolism, and signal transduction, affecting protein function and localization, since the O-linked N-acetylglucosamine moiety comes directly from the metabolism of glucose, lipids, and amino acids. The addition and removal of O-GlcNAc of target proteins are mediated by two highly conserved enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA), respectively. Deregulation of O-GlcNAcylation has been reported to be associated with various human diseases such as cancer, diabetes, and cardiovascular diseases. The contribution of deregulated O-GlcNAcylation to the progression and pathogenesis of NAFLD remains intriguing, and a better understanding of its roles in this pathophysiological context is required to uncover novel avenues for therapeutic intervention. By using a translational approach, our aim is to describe the role of OGT and O-GlcNAcylation in the pathogenesis of NAFLD. METHODS: We used primary mouse hepatocytes, human hepatic cell lines and in vivo mouse models of steatohepatitis to manipulate O-GlcNAc transferase (OGT). We also studied OGT and O-GlcNAcylation in liver samples from different cohorts of people with NAFLD. RESULTS: O-GlcNAcylation was upregulated in the liver of people and animal models with steatohepatitis. Downregulation of OGT in NAFLD-hepatocytes improved diet-induced liver injury in both in vivo and in vitro models. Proteomics studies revealed that mitochondrial proteins were hyper-O-GlcNAcylated in the liver of mice with steatohepatitis. Inhibition of OGT is able to restore mitochondrial oxidation and decrease hepatic lipid content in in vitro and in vivo models of NAFLD. CONCLUSIONS: These results demonstrate that deregulated hyper-O-GlcNAcylation favors NAFLD progression by reducing mitochondrial oxidation and promoting hepatic lipid accumulation.

Myeloid p38 activation maintains macrophage-liver crosstalk and BAT thermogenesis through IL-12-FGF21 axis.

Obesity features excessive fat accumulation in several body tissues and induces a state of chronic low-grade inflammation that contributes to the development of diabetes, steatosis, and insulin resistance. Recent research has shown that this chronic inflammation is crucially dependent on p38 pathway activity in macrophages, suggesting p38 inhibition as a possible treatment for obesity comorbidities. Nevertheless, we report here that lack of p38 activation in myeloid cells worsens high-fat diet-induced obesity, diabetes, and steatosis. Deficient p38 activation increases macrophage IL-12 production, leading to inhibition of hepatic FGF21 and reduction of thermogenesis in the brown fat. The implication of FGF21 in the phenotype was confirmed by its specific deletion in hepatocytes. We also found that IL-12 correlates with liver damage in human biopsies, indicating the translational potential of our results. Our findings suggest that myeloid p38 has a dual role in inflammation and that drugs targeting IL-12 might improve the homeostatic regulation of energy balance in response to metabolic stress.

Methionine adenosyltransferase 1a antisense oligonucleotides activate the liver-brown adipose tissue axis preventing obesity and associated hepatosteatosis.

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2-mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis.

High-performance nano-flow liquid chromatography column combined with high- and low-collision energy data-independent acquisition enables targeted and discovery identification of modified ribonucleotides by mass spectrometry.

Over 170 post-transcriptional RNA modifications have been described and are common in all kingdoms of life. These modifications range from methylation to complex chemical structures, with methylation being the most abundant. RNA modifications play a key role in RNA folding and function and their dysregulation in humans has been linked to several diseases such as cancer, metabolic diseases or neurological disorder. Nowadays, liquid chromatography-tandem mass spectrometry is considered the gold standard method for the identification and quantification of these modifications due to its sensitivity and accuracy. However, the analysis of modified ribonucleosides by mass spectrometry is complex due to the presence of positional isomers. In this scenario, optimal separation of these compounds by highly sensitive liquid chromatography combined with the generation of high-information spectra is critical to unequivocally identify them, especially in high-complex mixtures. Here we present an analytical method that comprises a new type of mixed-mode nano-flow liquid chromatography column combined with high- and low-collision energy data-independent mass spectrometric acquisition for the identification and quantitation of modified ribonucleosides. The method produces content-rich spectra and combines targeted and screening capabilities thus enabling the identification of a variety of modified nucleosides in biological matrices by single-shot liquid chromatographic analysis coupled to mass spectrometry.

O-GlcNAcylated p53 in the liver modulates hepatic glucose production.

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.

Human tRNAs with inosine 34 are essential to efficiently translate eukarya-specific low-complexity proteins.

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.

Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation.

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m6A), has been implicated in neurogenesis and memory formation. However, whether additional RNA modifications may be playing a role in neuronal functions and in response to environmental queues is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m2,2G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNAAla(AGC) isodecoders at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes may play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.

The L-α-Lysophosphatidylinositol/G Protein-Coupled Receptor 55 System Induces the Development of Nonalcoholic Steatosis and Steatohepatitis.

BACKGROUND AND AIMS: G protein-coupled receptor (GPR) 55 is a putative cannabinoid receptor, and l-α-lysophosphatidylinositol (LPI) is its only known endogenous ligand. Although GPR55 has been linked to energy homeostasis in different organs, its specific role in lipid metabolism in the liver and its contribution to the pathophysiology of nonalcoholic fatty liver disease (NAFLD) remains unknown. APPROACH AND RESULTS: We measured (1) GPR55 expression in the liver of patients with NAFLD compared with individuals without obesity and without liver disease, as well as animal models with steatosis and nonalcoholic steatohepatitis (NASH), and (2) the effects of LPI and genetic disruption of GPR55 in mice, human hepatocytes, and human hepatic stellate cells. Notably, we found that circulating LPI and liver expression of GPR55 were up-regulated in patients with NASH. LPI induced adenosine monophosphate-activated protein kinase activation of acetyl-coenzyme A carboxylase (ACC) and increased lipid content in human hepatocytes and in the liver of treated mice by inducing de novo lipogenesis and decreasing ß-oxidation. The inhibition of GPR55 and ACCα blocked the effects of LPI, and the in vivo knockdown of GPR55 was sufficient to improve liver damage in mice fed a high-fat diet and in mice fed a methionine-choline-deficient diet. Finally, LPI promoted the initiation of hepatic stellate cell activation by stimulating GPR55 and activation of ACC. CONCLUSIONS: The LPI/GPR55 system plays a role in the development of NAFLD and NASH by activating ACC.

Integrative analyses of the RNA modification machinery reveal tissue- and cancer-specific signatures.

BACKGROUND: RNA modifications play central roles in cellular fate and differentiation. However, the machinery responsible for placing, removing, and recognizing more than 170 RNA modifications remains largely uncharacterized and poorly annotated, and we currently lack integrative studies that identify which RNA modification-related proteins (RMPs) may be dysregulated in each cancer type. RESULTS: Here, we perform a comprehensive annotation and evolutionary analysis of human RMPs, as well as an integrative analysis of their expression patterns across 32 tissues, 10 species, and 13,358 paired tumor-normal human samples. Our analysis reveals an unanticipated heterogeneity of RMP expression patterns across mammalian tissues, with a vast proportion of duplicated enzymes displaying testis-specific expression, suggesting a key role for RNA modifications in sperm formation and possibly intergenerational inheritance. We uncover many RMPs that are dysregulated in various types of cancer, and whose expression levels are predictive of cancer progression. Surprisingly, we find that several commonly studied RNA modification enzymes such as METTL3 or FTO are not significantly upregulated in most cancer types, whereas several less-characterized RMPs, such as LAGE3 and HENMT1, are dysregulated in many cancers. CONCLUSIONS: Our analyses reveal an unanticipated heterogeneity in the expression patterns of RMPs across mammalian tissues and uncover a large proportion of dysregulated RMPs in multiple cancer types. We provide novel targets for future cancer research studies targeting the human epitranscriptome, as well as foundations to understand cell type-specific behaviors that are orchestrated by RNA modifications.

Accurate detection of m6A RNA modifications in native RNA sequences.

The epitranscriptomics field has undergone an enormous expansion in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here, we show that using direct RNA sequencing, N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Specifically, we find that our algorithm, trained with m6A-modified and unmodified synthetic sequences, can predict m6A RNA modifications with ~90% accuracy. We then extend our findings to yeast data sets, finding that our method can identify m6A RNA modifications in vivo with an accuracy of 87%. Moreover, we further validate our method by showing that these 'errors' are typically not observed in yeast ime4-knockout strains, which lack m6A modifications. Our results open avenues to investigate the biological roles of RNA modifications in their native RNA context.

A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS).

Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.

The RNA modification landscape in human disease.

RNA modifications have been historically considered as fine-tuning chemo-structural features of infrastructural RNAs, such as rRNAs, tRNAs, and snoRNAs. This view has changed dramatically in recent years, to a large extent as a result of systematic efforts to map and quantify various RNA modifications in a transcriptome-wide manner, revealing that RNA modifications are reversible, dynamically regulated, far more widespread than originally thought, and involved in major biological processes, including cell differentiation, sex determination, and stress responses. Here we summarize the state of knowledge and provide a catalog of RNA modifications and their links to neurological disorders, cancers, and other diseases. With the advent of direct RNA-sequencing technologies, we expect that this catalog will help prioritize those RNA modifications for transcriptome-wide maps.

Diagnostic value of core needle biopsy and fine-needle aspiration in salivary gland lesions.

BACKGROUND: Core needle biopsy (CNB) has gained acceptance as a minimally invasive procedure in the head and neck. Nevertheless, many concerns arise regarding the value and safety of this method in the assessment of salivary gland lesions. METHODS: This prospective study comprises 111 patients with a salivary gland lesion. The results of ultrasound-guided CNB were compared with those of fine-needle aspiration (FNA) in the 103 histologically verified cases. RESULTS: CNB achieved a higher accuracy than FNA in identifying true neoplasms (98% vs 91%) and detecting malignancy (99% vs 87%), and was also superior to FNA providing a specific diagnosis (93% vs 74%). In both methods, no complications, such as bleeding, infection, nerve injury, or tumor-cell seeding, occurred. CONCLUSION: CNB is a simple, safe, and highly accurate procedure, which should be considered as an additional diagnostic tool in the assessment of salivary gland lesions. © 2015 Wiley Periodicals, Inc. Head Neck 38: E346-E352, 2016.

Cooperation for Better Inhibiting.

Cladosporin is an antimalarial drug that acts as an ATP-mimetic to selectively inhibit Plasmodium lysyl-tRNA synthetase. Using multiple crystal structures, Fang et al. (2015) reveal in this issue of Chemistry & Biology the fascinating mechanism responsible for cladosporin selectivity.

Paediatric otogenic lateral sinus thrombosis: therapeutic management, outcome and thrombophilic evaluation.

OBJECTIVE: Otogenic lateral sinus thrombosis (LST) in children represents a serious condition with potential long-lasting morbidity. The role of adjunct anticoagulation therapy and the benefit of an analysis of prothrombotic factors are unclear. The aim of the study was to report therapeutic management and outcome, analyze prothrombotic factors in children with otogenic LST treated with mastoidectomy/antibiotics/anticoagulation and to evaluate the results with a review of the literature. METHODS: Retrospective chart review of 9 children with otogenic LST (2000-2009) and literature search in PubMed. RESULTS: The most frequent sign was fever in 88%, while neurologic findings were seen in 55%. Streptococci was the most common bacteria (55%). Prothrombotic factors were normal in all children. All patients received therapeutic anticoagulation, without experiencing bleeding complications. Eight children made a full recovery, neurologic sequelae persisted in one. The literature review of 115 children identified fever as the most prominent sign, reported the absence of neurologic findings in almost 50% of cases and confirmed the major role of streptococci. Anticoagulation, as adjunct therapy, was given to 38% of patients in the therapeutic range with a trend towards better neurologic outcome. A prothrombotic analysis was reported in 5 studies with positive results in 2. CONCLUSIONS: Surgery and antibiotics represent the mainstay of the therapy. Anticoagulation can be safely added in view of the high potential for morbidity and might reduce neurologic sequelae. Bacteria with thrombotic activity seem to be an important aetiology. In contrast, a prothrombotic disposition seems to play a minor role in the development of otogenic LST.

Selective inhibition of an apicoplastic aminoacyl-tRNA synthetase from Plasmodium falciparum.

The resistance of malaria parasites to available drugs continues to grow, and this makes the need for new antimalarial therapies pressing. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes and well-established antibacterial targets and so constitute a promising set of targets for the development of new antimalarials. Despite their potential as drug targets, apicoplastic ARSs remain unexplored. We have characterized the lysylation system of Plasmodium falciparum, and designed, synthesized, and tested a set of inhibitors based on the structure of the natural substrate intermediate: lysyl-adenylate. Here we demonstrate that selective inhibition of apicoplastic ARSs is feasible and describe new compounds that that specifically inhibit Plasmodium apicoplastic lysyl-tRNA synthetase and show antimalarial activities in the micromolar range.

Role of ultrasound-guided core-needle biopsy in the assessment of head and neck lesions: a meta-analysis and systematic review of the literature.

BACKGROUND: Core-needle biopsy (CNB) has occasionally been used in the work-up of head and neck lesions. However, no systematic review of this simple, minimally invasive method has yet been performed. METHODS: A systematic review of the literature and meta-analysis of data extracted from 16 included studies were performed. A total of 1291 cervical lesions in 1267 patients were examined by CNB. This resulted in 1232 adequate samples, from which 554 were subsequently confirmed by excisional biopsy. RESULTS: CNB was able to identify true neoplasms and detect malignancy in head and neck lesions with an overall accuracy of 94% and 96%, respectively, even though there was a significant difference between the histologically verified and all adequate samples. CNB provided a correct specific diagnosis in 87% of cases without major complications and achieved a higher accuracy than that of fine-needle aspiration in detecting malignancy. CONCLUSIONS: This meta-analysis confirms that CNB is an excellent method in the assessment of salivary gland lesions and lymphadenopathies inclusive of malignant lymphoma, but it is not ideal for evaluating thyroid lesions.