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In recent years, nanopore direct RNA sequencing (DRS) became a valuable tool for studying the epitranscriptome, due to its ability to detect multiple modifications within the same full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling 'errors' in DRS datasets, N6-methyladenosine (m6A) modifications produce relatively low 'errors' compared to other RNA modifications, limiting the applicability of this approach to m6A sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully unmodified sequences, increases the 'error'signal of m6A, leading to enhanced detection and improved sensitivity even at low stoichiometries. Moreover, we find that high-accuracy alternative RNA basecalling models can show up to 97% median basecalling accuracy, outperforming currently available RNA basecalling models, which show 91% median basecalling accuracy. Notably, the use of high-accuracy basecalling models is accompanied by a significant increase in the number of mapped reads -especially in shorter RNA fractions- and increased basecalling error signatures at pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) modified sites. Overall, our work demonstrates that alternative RNA basecalling models can be used to improve the detection of RNA modifications, read mappability, and basecalling accuracy in nanopore DRS datasets., (Published by Cold Spring Harbor Laboratory Press.)

Rationale: The study addresses the challenge of identifying RNA post-transcriptional modifications when commercial standards are not available to generate reference spectral libraries. It proposes employing homologous nucleobases and deoxyribonucleosides as alternative reference spectral libraries to aid in identifying modified ribonucleosides and distinguishing them from their positional isomers when the standards are unavailable., Methods: Complete sets of ribonucleoside, deoxyribonucleoside and nucleobase standards were analyzed using high-performance nano-flow liquid chromatography coupled to an Orbitrap Eclipse Tribrid mass spectrometer. Spectral libraries were constructed from homologous nucleobases and deoxyribonucleosides using targeted MS2 and neutral-loss-triggered MS3 methods, and collision energies were optimized. The feasibility of using these libraries for identifying modified ribonucleosides and their positional isomers was assessed through comparison of spectral fragmentation patterns., Results: Our analysis reveals that both MS2 and neutral-loss-triggered MS3 methods yielded rich spectra with similar fragmentation patterns across ribonucleosides, deoxyribonucleosides and nucleobases. Moreover, we demonstrate that spectra from nucleobases and deoxyribonucleosides, generated at optimized collision energies, exhibited sufficient similarity to those of modified ribonucleosides to enable their use as reference spectra for accurate identification of positional isomers within ribonucleoside families., Conclusions: The study demonstrates the efficacy of utilizing homologous nucleobases and deoxyribonucleosides as interchangeable reference spectral libraries for identifying modified ribonucleosides and their positional isomers. This approach offers a valuable solution for overcoming limitations posed by the unavailability of commercial standards, enhancing the analysis of RNA post-transcriptional modifications via mass spectrometry., (© 2024 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Down syndrome (DS) stands as the prevalent genetic cause of intellectual disability, yet comprehensive understanding of its cellular and molecular underpinnings remains limited. In this study, we explore the cellular landscape of the hippocampus in a DS mouse model, the Ts65Dn, through single-nuclei transcriptional profiling. Our findings demonstrate that trisomy manifests as a highly specific modification of the transcriptome within distinct cell types. Remarkably, we observed a significant shift in the transcriptomic profile of granule cells in the dentate gyrus (DG) associated with trisomy. We identified the downregulation of a specific small nucleolar RNA host gene, Snhg11, as the primary driver behind this observed shift in the trisomic DG. Notably, reduced levels of Snhg11 in this region were also observed in a distinct DS mouse model, the Dp(16)1Yey, as well as in human postmortem brain tissue, indicating its relevance in Down syndrome. To elucidate the function of this long non-coding RNA (lncRNA), we knocked down Snhg11 in the DG of wild-type mice. Intriguingly, this intervention alone was sufficient to impair synaptic plasticity and adult neurogenesis, resembling the cognitive phenotypes associated with trisomy in the hippocampus. Our study uncovers the functional role of Snhg11 in the DG and underscores the significance of this lncRNA in intellectual disability. Furthermore, our findings highlight the importance of DG in the memory deficits observed in Down syndrome., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Despite the nuclear localization of the m 6 A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m 6 A-modified. However, these findings are mostly based on m 6 A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m 6 A in CHIKV and DENV RNAs. For this, we combine m 6 A-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 m 6 A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m 6 A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m 6 A machinery's localization. Our results challenge the prevailing notion that m 6 A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches., (© 2024. The Author(s).)

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress., (© 2023. The Author(s).)

The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse 'specialized ribosomes' is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralogue of RPL3 (uL3) that is exclusively expressed in skeletal muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon RPL3L depletion, RPL3 becomes up-regulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L modulates neither translational efficiency nor ribosome affinity towards a specific subset of transcripts. In contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of fine-tuning of mitochondrial activity. Our results demonstrate that the existence of tissue-specific RP paralogues does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome., (© 2022. The Author(s).)

This chapter describes MasterOfPores v.2 (MoP2), an open-source suite of pipelines for processing and analyzing direct RNA Oxford Nanopore sequencing data. The MoP2 relies on the Nextflow DSL2 framework and Linux containers, thus enabling reproducible data analysis in transcriptomic and epitranscriptomic studies. We introduce the key concepts of MoP2 and provide a step-by-step fully reproducible and complete example of how to use the workflow for the analysis of S. cerevisiae total RNA samples sequenced using MinION flowcells. The workflow starts with the pre-processing of raw FAST5 files, which includes basecalling, read quality control, demultiplexing, filtering, mapping, estimation of per-gene/transcript abundances, and transcriptome assembly, with support of the GPU computing for the basecalling and read demultiplexing steps. The secondary analyses of the workflow focus on the estimation of RNA poly(A) tail lengths and the identification of RNA modifications. The MoP2 code is available at https://github.com/biocorecrg/MOP2 and is distributed under the MIT license., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Chemical RNA modifications, collectively referred to as the "epitranscriptome," are essential players in fine-tuning gene expression. Our ability to analyze RNA modifications has improved rapidly in recent years, largely due to the advent of high-throughput sequencing methodologies, which typically consist of coupling modification-specific reagents, such as antibodies or enzymes, to next-generation sequencing. Recently, it also became possible to map RNA modifications directly by sequencing native RNAs using nanopore technologies, which has been applied for the detection of a number of RNA modifications, such as N6-methyladenosine (m 6 A), pseudouridine (Ψ), and inosine (I). However, the signal modulations caused by most RNA modifications are yet to be determined. A global effort is needed to determine the signatures of the full range of RNA modifications to avoid the technical biases that have so far limited our understanding of the epitranscriptome., (© 2022 Begik et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Ample evidence indicates that codon usage bias regulates gene expression. How viruses, such as the emerging mosquito-borne Chikungunya virus (CHIKV), express their genomes at high levels despite an enrichment in rare codons remains a puzzling question. Using ribosome footprinting, we analyze translational changes that occur upon CHIKV infection. We show that CHIKV infection induces codon-specific reprogramming of the host translation machinery to favor the translation of viral RNA genomes over host mRNAs with an otherwise optimal codon usage. This reprogramming was mostly apparent at the endoplasmic reticulum, where CHIKV RNAs show high ribosome occupancy. Mechanistically, it involves CHIKV-induced overexpression of KIAA1456, an enzyme that modifies the wobble U34 position in the anticodon of tRNAs, which is required for proper decoding of codons that are highly enriched in CHIKV RNAs. Our findings demonstrate an unprecedented interplay of viruses with the host tRNA epitranscriptome to adapt the host translation machinery to viral production., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Motivation: DNA and RNA modifications can now be identified using nanopore sequencing. However, we currently lack a flexible software to efficiently encode, store, analyze and visualize DNA and RNA modification data., Results: Here, we present ModPhred, a versatile toolkit that facilitates DNA and RNA modification analysis from nanopore sequencing reads in a user-friendly manner. ModPhred integrates probabilistic DNA and RNA modification information within the FASTQ and BAM file formats, can be used to encode multiple types of modifications simultaneously, and its output can be easily coupled to genomic track viewers, facilitating the visualization and analysis of DNA and RNA modification information in individual reads in a simple and computationally efficient manner., Availability and Implementation: ModPhred is available at https://github.com/novoalab/modPhred, is implemented in Python3, and is released under an MIT license. Docker images with all dependencies preinstalled are also provided., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m 6 A), 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 (m 2,2 G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNA Ala (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 covalent modification of RNA molecules is a pervasive feature of all classes of RNAs and has fundamental roles in the regulation of several cellular processes. Mapping the location of RNA modifications transcriptome-wide is key to unveiling their role and dynamic behaviour, but technical limitations have often hampered these efforts. Nanopore direct RNA sequencing is a third-generation sequencing technology that allows the sequencing of native RNA molecules, thus providing a direct way to detect modifications at single-molecule resolution. Despite recent advances, the analysis of nanopore sequencing data for RNA modification detection is still a complex task that presents many challenges. Many works have addressed this task using different approaches, resulting in a large number of tools with different features and performances. Here we review the diverse approaches proposed so far and outline the principles underlying currently available algorithms.

Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N 6 -methyladenosine, we show that other modifications, in particular pseudouridine (Ψ) and 2'-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on Ψ modification sites, we detected known and uncovered previously unreported Ψ sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent Ψ modification in yeast mitochondrial rRNA. To explore the dynamics of pseudouridylation, we treated yeast cells with oxidative, cold and heat stresses and detected heat-sensitive Ψ-modified sites in small nuclear RNAs, small nucleolar RNAs and mRNAs. Finally, we developed a software, nanoRMS, that estimates per-site modification stoichiometries by identifying single-molecule reads with altered current intensity and trace profiles. This work demonstrates that Nm and Ψ RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5' UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5' UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5' UTRs. A total of ~12,000 5' UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5' UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5' UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

RNA modifications play pivotal roles in the RNA life cycle and RNA fate, and are now appreciated as a major posttranscriptional regulatory layer in the cell. In the last few years, direct RNA nanopore sequencing (dRNA-seq) has emerged as a promising technology that can provide single-molecule resolution maps of RNA modifications in their native RNA context. While native RNA can be successfully sequenced using this technology, the detection of RNA modifications is still challenging. Here, we provide an upgraded version of EpiNano (version 1.2), an algorithm to predict m 6 A RNA modifications from dRNA-seq datasets. The latest version of EpiNano contains models for predicting m 6 A RNA modifications in dRNA-seq data that has been base-called with Guppy. Moreover, it can now train models with features extracted from both base-called dRNA-seq FASTQ data and raw FAST5 nanopore outputs. Finally, we describe how EpiNano can be used in stand-alone mode to extract base-calling "error" features and current intensity information from dRNA-seq datasets. In this chapter, we provide step-by-step instructions on how to produce in vitro transcribed constructs to train EpiNano, as well as detailed information on how to use EpiNano to train, test, and predict m 6 A RNA modifications in dRNA-seq data.

Nanopore sequencing enables direct measurement of RNA molecules without conversion to cDNA, thus opening the gates to a new era for RNA biology. However, the lack of molecular barcoding of direct RNA nanopore sequencing data sets severely affects the applicability of this technology to biological samples, where RNA availability is often limited. Here, we provide the first experimental protocol and associated algorithm to barcode and demultiplex direct RNA nanopore sequencing data sets. Specifically, we present a novel and robust approach to accurately classify raw nanopore signal data by transforming current intensities into images or arrays of pixels, followed by classification using a deep learning algorithm. We demonstrate the power of this strategy by developing the first experimental protocol for barcoding and demultiplexing direct RNA sequencing libraries. Our method, DeePlexiCon, can classify 93% of reads with 95.1% accuracy or 60% of reads with 99.9% accuracy. The availability of an efficient and simple multiplexing strategy for native RNA sequencing will improve the cost-effectiveness of this technology, as well as facilitate the analysis of lower-input biological samples. Overall, our work exemplifies the power, simplicity, and robustness of signal-to-image conversion for nanopore data analysis using deep learning., (© 2020 Smith et al.; Published by Cold Spring Harbor Laboratory Press.)

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.

The direct RNA sequencing platform offered by Oxford Nanopore Technologies allows for direct measurement of RNA molecules without the need of conversion to complementary DNA, fragmentation or amplification. As such, it is virtually capable of detecting any given RNA modification present in the molecule that is being sequenced, as well as provide polyA tail length estimations at the level of individual RNA molecules. Although this technology has been publicly available since 2017, the complexity of the raw Nanopore data, together with the lack of systematic and reproducible pipelines, have greatly hindered the access of this technology to the general user. Here we address this problem by providing a fully benchmarked workflow for the analysis of direct RNA sequencing reads, termed MasterOfPores . The pipeline starts with a pre-processing module, which converts raw current intensities into multiple types of processed data including FASTQ and BAM, providing metrics of the quality of the run, quality-filtering, demultiplexing, base-calling and mapping. In a second step, the pipeline performs downstream analyses of the mapped reads, including prediction of RNA modifications and estimation of polyA tail lengths. Four direct RNA MinION sequencing runs can be fully processed and analyzed in 10 h on 100 CPUs. The pipeline can also be executed in GPU locally or in the cloud, decreasing the run time fourfold. The software is written using the NextFlow framework for parallelization and portability, and relies on Linux containers such as Docker and Singularity for achieving better reproducibility. The MasterOfPores workflow can be executed on any Unix-compatible OS on a computer, cluster or cloud without the need of installing any additional software or dependencies, and is freely available in Github (https://github.com/biocorecrg/master_of_pores). This workflow simplifies direct RNA sequencing data analyses, facilitating the study of the (epi)transcriptome at single molecule resolution., (Copyright © 2020 Cozzuto, Liu, Pryszcz, Pulido, Delgado-Tejedor, Ponomarenko and Novoa.)

Because of the degeneracy of the genetic code, multiple codons are translated into the same amino acid. Despite being "synonymous," these codons are not equally used. Selective pressures are thought to drive the choice among synonymous codons within a genome, while GC content, which is typically attributed to mutational drift, is the major determinant of variation across species. Here, we find that in addition to GC content, interspecies codon usage signatures can also be detected. More specifically, we show that a single amino acid, arginine, is the major contributor to codon usage bias differences across domains of life. We then exploit this finding and show that domain-specific codon bias signatures can be used to classify a given sequence into its corresponding domain of life with high accuracy. We then wondered whether the inclusion of codon usage codon autocorrelation patterns, which reflects the nonrandom distribution of codon occurrences throughout a transcript, might improve the classification performance of our algorithm. However, we find that autocorrelation patterns are not domain-specific, and surprisingly, are unrelated to tRNA reusage, in contrast to previous reports. Instead, our results suggest that codon autocorrelation patterns are a by-product of codon optimality throughout a sequence, where highly expressed genes display autocorrelated "optimal" codons, whereas lowly expressed genes display autocorrelated "nonoptimal" codons., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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, N 6 -methyladenosine (m 6 A) 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 m 6 A-modified and unmodified synthetic sequences, can predict m 6 A RNA modifications with ~90% accuracy. We then extend our findings to yeast data sets, finding that our method can identify m 6 A 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 m 6 A modifications. Our results open avenues to investigate the biological roles of RNA modifications in their native RNA context.

The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a central reaction in biology. Multiple regulatory pathways use the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here, we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated in arthropods a paralog protein (SLIMP) that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM and preventing mitochondrial DNA (mtDNA) accumulation. Thus, mitochondrial translation is directly coupled to mtDNA levels by a network based upon a profound structural modification of an animal ARS., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA folding plays a crucial role in RNA function. However, knowledge of the global structure of the transcriptome is limited to cellular systems at steady state, thus hindering the understanding of RNA structure dynamics during biological transitions and how it influences gene function. Here, we characterized mRNA structure dynamics during zebrafish development. We observed that on a global level, translation guides structure rather than structure guiding translation. We detected a decrease in structure in translated regions and identified the ribosome as a major remodeler of RNA structure in vivo. In contrast, we found that 3' untranslated regions (UTRs) form highly folded structures in vivo, which can affect gene expression by modulating microRNA activity. Furthermore, dynamic 3'-UTR structures contain RNA-decay elements, such as the regulatory elements in nanog and ccna1, two genes encoding key maternal factors orchestrating the maternal-to-zygotic transition. These results reveal a central role of RNA structure dynamics in gene regulatory programs.

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., (© 2017 Jonkhout et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA modifications can alter RNA structure-function relationships and various cellular processes. However, the genomic distribution and biological roles of most RNA modifications remain uncharacterized. Here, we propose using phage display antibody technology and direct sequencing through nanopores to facilitate systematic interrogation of the distribution, location and dynamics of RNA modifications.

Motivation: Riboswitches are cis-regulatory elements in mRNA, mostly found in Bacteria, which exhibit two main secondary structure conformations. Although one of them prevents the gene from being expressed, the other conformation allows its expression, and this switching process is typically driven by the presence of a specific ligand. Although there are a handful of known riboswitches, our knowledge in this field has been greatly limited due to our inability to identify their alternate structures from their sequences. Indeed, current methods are not able to predict the presence of the two functionally distinct conformations just from the knowledge of the plain RNA nucleotide sequence. Whether this would be possible, for which cases, and what prediction accuracy can be achieved, are currently open questions., Results: Here we show that the two alternate secondary structures of riboswitches can be accurately predicted once the 'switching sequence' of the riboswitch has been properly identified. The proposed SwiSpot approach is capable of identifying the switching sequence inside a putative, complete riboswitch sequence, on the basis of pairing behaviors, which are evaluated on proper sets of configurations. Moreover, it is able to model the switching behavior of riboswitches whose generated ensemble covers both alternate configurations. Beyond structural predictions, the approach can also be paired to homology-based riboswitch searches., Availability and Implementation: SwiSpot software, along with the reference dataset files, is available at: http://www.iet.unipi.it/a.bechini/swispot/Supplementary information: Supplementary data are available at Bioinformatics online., Contact: a.bechini@ing.unipi.it., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. The selective forces exerted by species-specific requirements and environmental conditions potentially shape quality-control mechanisms that serve to prevent mistranslation. A family of editing factors that are homologous to the editing domain of bacterial prolyl-tRNA synthetase includes the previously characterized trans-editing factors ProXp-ala and YbaK, which clear Ala-tRNA(Pro) and Cys-tRNA(Pro), respectively, and three additional homologs of unknown function, ProXp-x, ProXp-y, and ProXp-z. We performed an in vivo screen of 230 conditions in which an Escherichia coli proXp-y deletion strain was grown in the presence of elevated levels of amino acids and specific ARSs. This screen, together with the results of in vitro deacylation assays, revealed Ser- and Thr-tRNA deacylase function for this homolog. A similar activity was demonstrated for Bordetella parapertussis ProXp-z in vitro. These proteins, now renamed "ProXp-ST1" and "ProXp-ST2," respectively, recognize multiple tRNAs as substrates. Taken together, our data suggest that these free-standing editing domains have the ability to prevent mistranslation errors caused by a number of ARSs, including lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-tRNA synthetase, and alanyl-tRNA synthetase. The expression of these multifunctional enzymes is likely to provide a selective growth advantage to organisms subjected to environmental stresses and other conditions that alter the amino acid pool.

Accurate protein synthesis requires the hydrolytic editing of tRNAs incorrectly aminoacylated by aminoacyl-tRNA synthetases (ARSs). Recognition of cognate tRNAs by ARS is less error-prone than amino acid recognition, and, consequently, editing domains are generally believed to act only on the tRNAs cognate to their related ARSs. For example, the AlaX family of editing domains, including the editing domain of alanyl-tRNA synthetase and the related free-standing trans-editing AlaX enzymes, are thought to specifically act on tRNA(Ala), whereas the editing domains of threonyl-tRNA synthetases are specific for tRNA(Thr). Here we show that, contrary to this belief, AlaX-S, the smallest of the extant AlaX enzymes, deacylates Ser-tRNA(Thr) in addition to Ser-tRNA(Ala) and that a single residue is important to determine this behavior. Our data indicate that promiscuous forms of AlaX are ancestral to tRNA-specific AlaXs. We propose that former AlaX domains were used to maintain translational fidelity in earlier stages of genetic code evolution when mis-serylation of several tRNAs was possible., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts., (Copyright © 2015, American Association for the Advancement of Science.)

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

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., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Codon usage and tRNA abundance are critical parameters for gene synthesis. However, the forces determining codon usage bias within genomes and between organisms, as well as the functional roles of biased codon compositions, remain poorly understood. Similarly, the composition and dynamics of mature tRNA populations in cells in terms of isoacceptor abundances, and the prevalence and function of base modifications are not well understood. As we begin to decipher some of the rules that govern codon usage and tRNA abundances, it is becoming clear that these parameters are a way to not only increase gene expression, but also regulate the speed of ribosomal translation, the efficiency of protein folding, and the coordinated expression of functionally related gene families. Here, we discuss the importance of codon-anticodon interactions in translation regulation and highlight the contribution of non-random codon distributions and post-transcriptional base modifications to this regulation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Transfer RNA (tRNA) gene content is a differentiating feature of genomes that contributes to the efficiency of the translational apparatus, but the principles shaping tRNA gene copy number and codon composition are poorly understood. Here, we report that the emergence of two specific tRNA modifications shaped the structure and composition of all extant genomes. Through the analysis of more than 500 genomes, we identify two kingdom-specific tRNA modifications as major contributors that separated archaeal, bacterial, and eukaryal genomes in terms of their tRNA gene composition. We show that, contrary to prior observations, genomic codon usage and tRNA gene frequencies correlate in all kingdoms if these two modifications are taken into account and that presence or absence of these modifications explains patterns of gene expression observed in previous studies. Finally, we experimentally demonstrate that human gene expression levels correlate well with genomic codon composition if these identified modifications are considered., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We present here a systematic exploration of the quality of protein structures derived from homology modeling when used as templates for high-throughput docking. It is found that structures derived from homology modeling are often similar in quality for docking purposes than real crystal structures, even in cases where the template used to create the structural model shows only a moderate sequence identity with the protein of interest. We designed an "ensemble docking" approach based on the use of multiple homology models. The method provides results which are usually of better quality than those expected from single experimental X-ray structures. The use of this approach allows us to increase around five times the universe of use of high-throughput docking approaches for human proteins, by covering over 75% of known human therapeutic targets.

During their extended evolution genes coding for aminoacyl-tRNA synthetases (ARS) have experienced numerous instances of duplication, insertion and deletion of domains. The ARS-related proteins that have resulted from these genetic events are generally known as aminoacyl-tRNA synthetase-like proteins (ARS-like). This heterogeneous group of polypeptides carries out an equally varied number of functions that need not be related to gene translation. Several of these proteins remain uncharacterized. At least 16 different ARS-like proteins have been identified to date, but their functions remain incompletely understood. Here we review the individual phylogenetic distribution of these proteins in bacteria, and apply a new genomics method to determine their potential implication in pathogenicity.