ORCID Profile
0000-0002-9367-6311
Current Organisation
Centre for Genomic Regulation
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Genomics | Genetics | Epigenetics (incl. Genome Methylation and Epigenomics) | Genome Structure and Regulation | Bioinformatics | Systems Biology |
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Human Biological Preventatives (e.g. Vaccines) | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in Technology
Related Links
Publications
ModPhred: an integrative toolkit for the analysis and storage of nanopore sequencing DNA and RNA modification data
Publisher: Oxford University Press (OUP)
Date: 22-07-2021
DOI: 10.1093/BIOINFORMATICS/BTAB539
Abstract: 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. 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 in idual reads in a simple and computationally efficient manner. ModPhred is available at ovoalab/modPhred, is implemented in Python3, and is released under an MIT license. Docker images with all dependencies preinstalled are also provided. Supplementary data are available at Bioinformatics online.
A genomics method to identify pathogenicity‐related proteins. Application to aminoacyl‐tRNA synthetase‐like proteins
Publisher: Wiley
Date: 12-11-2009
DOI: 10.1016/J.FEBSLET.2009.11.016
Abstract: 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 in idual phylogenetic distribution of these proteins in bacteria, and apply a new genomics method to determine their potential implication in pathogenicity.
Ancestral AlaX Editing Enzymes for Control of Genetic Code Fidelity Are Not tRNA-specific
Publisher: Elsevier BV
Date: 04-2015
Ensemble Docking from Homology Models
Publisher: American Chemical Society (ACS)
Date: 16-07-2010
DOI: 10.1021/CT100246Y
Abstract: 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.
Quantitative profiling of native RNA modifications and their dynamics using nanopore sequencing
Publisher: Cold Spring Harbor Laboratory
Date: 06-07-2020
DOI: 10.1101/2020.07.06.189969
Abstract: A broad ersity of modifications decorate RNA molecules. Originally conceived as static components, evidence is accumulating that some RNA modifications may be dynamic, contributing to cellular responses to external signals and environmental circumstances. A major difficulty in studying these modifications, however, is the need of tailored protocols to map each modification type in idually. Here, we present a new approach that uses direct RNA nanopore sequencing to identify and quantify RNA modifications present in native RNA molecules. First, we show that each RNA modification type results in a distinct and characteristic base-calling ‘error’ signature, which we validate using a battery of genetic strains lacking either pseudouridine (Y) or 2’-O-methylation (Nm) modifications. We then demonstrate the value of these signatures for de novo prediction of Y modifications transcriptome-wide, confirming known Y-modified sites as well as uncovering novel Y sites in mRNAs, ncRNAs and rRNAs, including a previously unreported Pus4-dependent Y modification in yeast mitochondrial rRNA, which we validate using orthogonal methods. To explore the dynamics of pseudouridylation across environmental stresses, we treat the cells with oxidative, cold and heat stresses, finding that yeast ribosomal rRNA modifications do not change upon environmental exposures, contrary to the general belief. By contrast, our method reveals many novel heat-sensitive Y-modified sites in snRNAs, snoRNAs and mRNAs, in addition to recovering previously reported sites. Finally, we develop a novel software, nanoRMS , which we show can estimate per-site modification stoichiometries from in idual RNA molecules by identifying the reads with altered current intensity and trace profiles, and quantify the RNA modification stoichiometry changes between two conditions. Our work demonstrates that Y RNA modifications can be predicted de novo and in a quantitative manner using native RNA nanopore sequencing.
Saturation of recognition elements blocks evolution of new tRNA identities
Publisher: American Association for the Advancement of Science (AAAS)
Date: 04-2016
Abstract: The size of the genetic code is limited by the ability of transfer RNAs to acquire new identities.
MasterOfPores: A Workflow for the Analysis of Oxford Nanopore Direct RNA Sequencing Datasets
Publisher: Frontiers Media SA
Date: 17-03-2020
Accurate detection of m6A RNA modifications in native RNA sequences
Publisher: Springer Science and Business Media LLC
Date: 09-09-2019
DOI: 10.1038/S41467-019-11713-9
Abstract: 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.
Integrative analyses of the RNA modification machinery reveal tissue- and cancer-specific signatures
Publisher: Springer Science and Business Media LLC
Date: 07-05-2020
DOI: 10.1186/S13059-020-02009-Z
Abstract: 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. 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 s les. 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. 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.
Native RNA nanopore sequencing reveals antibiotic-induced loss of rRNA modifications in the A- and P-sites
Publisher: Cold Spring Harbor Laboratory
Date: 21-03-2023
DOI: 10.1101/2023.03.21.533606
Abstract: The biological relevance and dynamics of mRNA modifications have been extensively studied in the past few years, revealing their key roles in major cellular processes, such as cellular differentiation or sex determination. However, whether rRNA modifications are dynamically regulated, and under which conditions, remains largely unclear. Here, we performed a systematic characterization of bacterial rRNA modification dynamics upon exposure to erse antibiotics using native RNA nanopore sequencing. To identify significant rRNA modification changes, we developed NanoConsensus , a novel pipeline that integrates the estimates from multiple RNA modification detection algorithms, predicting differentially modified rRNA sites with very low false positive rates and high replicability. We showed that NanoConsensus is robust across RNA modification types, stoichiometries and coverage, and outperforms all in idual algorithms tested. Using this approach, we identified multiple rRNA modifications that are lost upon the presence of antibiotics, showing that rRNA modification profiles are altered in an antibiotic-specific manner. We found that significantly altered rRNA modified sites upon antibiotic exposure are located in the vicinity of the A and P-sites of the ribosome, possibly contributing to antibiotic resistance. We then systematically examined whether loss of ‘antibiotic-sensitive’ rRNA modifications may be sufficient to confer antibiotic resistance, finding that depletion of some rRNA modification enzymes guiding dysregulated rRNA modifications confers increased antibiotic resistance. Altogether, our work reveals that rRNA modification profiles can be rapidly altered in response to environmental exposures, and that nanopore sequencing can accurately identify dysregulated rRNA modifications, contributing to the mechanistic dissection of antibiotic resistance. Moreover, we provide a novel, robust workflow to study rRNA modification dynamics in any species using nanopore sequencing in a scalable and reproducible manner.
The RNA modification landscape in human disease
Publisher: Cold Spring Harbor Laboratory
Date: 30-08-2017
Abstract: 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.
Molecular barcoding of native RNAs using nanopore sequencing and deep learning
Publisher: Cold Spring Harbor Laboratory
Date: 09-2020
Abstract: 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 s les, 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 s les. Overall, our work exemplifies the power, simplicity, and robustness of signal-to-image conversion for nanopore data analysis using deep learning.
Cooperation for Better Inhibiting
Publisher: Elsevier BV
Date: 06-2015
DOI: 10.1016/J.CHEMBIOL.2015.06.002
Abstract: 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.
Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation
Publisher: Informa UK Limited
Date: 15-02-2021
A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS)
Publisher: Springer Science and Business Media LLC
Date: 28-06-2019
DOI: 10.1038/S41467-019-10912-8
Abstract: 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.
Homologous trans -editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation
Publisher: Proceedings of the National Academy of Sciences
Date: 27-04-2015
Abstract: A key step in protein synthesis is catalyzed by aminoacyl-tRNA synthetases (ARSs), which attach specific amino acids to tRNAs. Errors caused by ARS amino acid misactivation require proofreading activities encoded in editing domains. Approximately half of the ARSs possess intrinsic editing functions, and single-domain editing proteins also are encoded in some organisms. The present study used an in vivo screen coupled with in vitro assays to elucidate the substrate specificity of two free-standing editing domains, revealing that they are multifunctional Ser/Thr-tRNA deacylases with the ability to prevent errors caused by a number of ARSs. Trans -editing proteins with broad substrate specificities provide a growth advantage to microbes under conditions in which amino acid pools are altered and may represent novel targets.
Dynamic interplay between RPL3- and RPL3L-containing ribosomes modulates mitochondrial activity in the mammalian heart
Publisher: Oxford University Press (OUP)
Date: 07-03-2023
DOI: 10.1093/NAR/GKAD121
Abstract: The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally erse ‘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.
Exploring the epitranscriptome by native RNA sequencing
Publisher: Cold Spring Harbor Laboratory
Date: 14-09-2022
Abstract: 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.
Computational methods for RNA modification detection from nanopore direct RNA sequencing data
Publisher: Informa UK Limited
Date: 24-09-2021
Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo
Publisher: Proceedings of the National Academy of Sciences
Date: 08-12-2014
Abstract: Malaria remains one of the main health threats in the developing world, with staggering social and economic costs. Resistance to artemisins, the main pharmacological tool currently available against malaria, has been widely reported. Borrelidin, a natural compound that inhibits threonyl-tRNA synthetase, has long been studied for its antibacterial and antiparasitic properties, but undesirable toxic effects prevented its further clinical development. Here we present a group of borrelidin derivatives that retain their ability to inhibit Plasmodium threonyl-tRNA synthetase but not its human homolog. Furthermore, we demonstrate, for the first time to our knowledge, that these compounds are capable of effectively clearing a Plasmodium infection in animals, curing malaria with a potency equivalent to reference drugs such as chloroquine.
Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing
Publisher: Springer Science and Business Media LLC
Date: 06-04-2023
DOI: 10.1038/S41587-023-01743-6
Abstract: 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.
Charting the unknown epitranscriptome
Publisher: Springer Science and Business Media LLC
Date: 10-05-2017
DOI: 10.1038/NRM.2017.49
Abstract: 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.
Machine-Type Communication with Random Access and Data Aggregation: A Stochastic Geometry Approach
Publisher: IEEE
Date: 12-2018
Speeding with control: codon usage, tRNAs, and ribosomes
Publisher: Elsevier BV
Date: 11-2012
DOI: 10.1016/J.TIG.2012.07.006
Abstract: 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.
Long-read sequencing in the era of epigenomics and epitranscriptomics
Publisher: Springer Science and Business Media LLC
Date: 2023
Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing
Publisher: Springer Science and Business Media LLC
Date: 19-12-2022
DOI: 10.1038/S41592-022-01714-W
Abstract: 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 lification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide ersity 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 in idual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
Elucidation of Codon Usage Signatures across the Domains of Life
Publisher: Oxford University Press (OUP)
Date: 20-05-2019
Abstract: 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.
Comprehensive map of ribosomal 2′-O-methylation and C/D box snoRNAs inDrosophila melanogaster
Publisher: Cold Spring Harbor Laboratory
Date: 25-05-2023
DOI: 10.1101/2023.05.25.542231
Abstract: 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.
CHIKV infection reprograms codon optimality to favor viral RNA translation by altering the tRNA epitranscriptome
Publisher: Springer Science and Business Media LLC
Date: 11-08-2022
DOI: 10.1038/S41467-022-31835-X
Abstract: 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.
N6-methyladenosine modification is not a general trait of viral RNA genomes
Publisher: Cold Spring Harbor Laboratory
Date: 13-10-2023
Highly evolvable malaria vectors: The genomes of 16 Anopheles mosquitoes
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-01-2015
Abstract: Control of mosquito vectors has historically proven to be an effective means of eliminating malaria. Human malaria is transmitted only by mosquitoes in the genus Anopheles , but not all species within the genus, or even all members of each vector species, are efficient malaria vectors. Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. This variation in vectorial capacity suggests an underlying genetic/genomic plasticity that results in variation of key traits determining vectorial capacity within the genus. Sequencing the genome of Anopheles gambiae , the most important malaria vector in sub-Saharan Africa, has offered numerous insights into how that species became highly specialized to live among and feed upon humans and how susceptibility to mosquito control strategies is determined. Until very recently, similar genomic resources have not existed for other anophelines, limiting comparisons to in idual genes or sets of genomic markers with no genome-wide data to investigate attributes associated with vectorial capacity across the genus. We sequenced and assembled the genomes and transcriptomes of 16 anophelines from Africa, Asia, Europe, and Latin America, spanning ~100 million years of evolution and chosen to represent a range of evolutionary distances from An. gambiae , a variety of geographic locations and ecological conditions, and varying degrees of vectorial capacity. Genome assembly quality reflected DNA template quality and homozygosity. Despite variation in contiguity, the assemblies were remarkably complete and searches for arthropod-wide single-copy orthologs generally revealed few missing genes. Genome annotation supported with RNA sequencing transcriptomes yielded between 10,738 and 16,149 protein-coding genes for each species. Relative to Drosophila, the closest dipteran genus for which equivalent genomic resources exist, Anopheles exhibits a dynamic genomic evolutionary profile. Comparative analyses show a fivefold faster rate of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses in Anopheles . Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead ersify through protein-sequence changes. We also document evidence of variation in important reproductive phenotypes, genes controlling immunity to Plasmodium malaria parasites and other microbes, genes encoding cuticular and salivary proteins, and genes conferring metabolic insecticide resistance. 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. Anopheline mosquitoes exhibit a molecular evolutionary profile very distinct from Drosophila , and their genomes harbor strong evidence of functional variation in traits that determine vectorial capacity. These 16 new reference genome assemblies provide a foundation for hypothesis generation and testing to further our understanding of the erse biological traits that determine vectorial capacity. The maximum likelihood molecular phylogeny of all sequenced anophelines and two mosquito outgroups was constructed from the aligned protein sequences of 1085 single-copy orthologs. Shapes between branch termini and species names indicate vector status and are colored according to geographic ranges depicted on the map.
Selective Inhibition of an Apicoplastic Aminoacyl‐tRNA Synthetase from Plasmodium falciparum
Publisher: Wiley
Date: 27-02-2013
Abstract: 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.
A Role for tRNA Modifications in Genome Structure and Codon Usage
Publisher: Elsevier BV
Date: 03-2012
DOI: 10.1016/J.CELL.2012.01.050
Abstract: 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.
Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing
Publisher: Springer Science and Business Media LLC
Date: 13-05-2021
DOI: 10.1038/S41587-021-00915-6
Abstract: 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
High-Throughput 5’ UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies
Publisher: Cold Spring Harbor Laboratory
Date: 25-03-2020
DOI: 10.1101/2020.03.24.006486
Abstract: Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5’ untranslated regions (5’ UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5’UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5’ UTRs with high translation efficiencies and used this information with in silico genetic algorithms to generate synthetic 5’ UTRs. A total of ∼12,000 5’ UTRs were 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 identified three synthetic 5’ UTRs that outperformed commonly used non-viral gene therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5’ UTRs enabled even higher protein expression than obtained with each in idual 5’ UTR. 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.
Best practices for genome-wide RNA structure analysis: combination of mutational profiles and drop-off information
Publisher: Cold Spring Harbor Laboratory
Date: 21-08-2017
DOI: 10.1101/176883
Abstract: Genome-wide RNA structure maps have recently become available through the coupling of in vivo chemical probing reagents with next-generation sequencing. Initial analyses relied on the identification of truncated reverse transcription reads to identify the chemically modified nucleotides, but recent studies have shown that mutational signatures can also be used. While these two methods have been employed interchangeably, here we show that they actually provide complementary information. Consequently, analyses using exclusively one of the two methodologies may disregard a significant portion of the structural information. We also show that the identity and sequence environment of the modified nucleotide greatly affect the odds of introducing a mismatch or causing reverse transcriptase drop-off. Finally, we identify specific mismatch signatures generated by dimethyl sulfate probing that can be exploited to remove false positives typically produced in RNA structurome analyses, and how these signatures vary depending on the reverse transcription enzyme used.
Analyses of mRNA structure dynamics identify embryonic gene regulatory programs.
Publisher: No publisher found
Date: 2018
Dynamic interplay between RPL3- and RPL3L-containing ribosomes modulates mitochondrial activity in the mammalian heart
Publisher: Cold Spring Harbor Laboratory
Date: 04-12-2021
DOI: 10.1101/2021.12.04.471171
Abstract: The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally erse ‘specialized ribosomes’ is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralog of RPL3 (uL3) that is exclusively expressed in 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 upregulated, 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 neither modulates translational efficiency nor ribosome affinity towards a specific subset of transcripts. By 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 mitochondrial activity fine-tuning. Our results demonstrate that the existence of tissue-specific RP paralogs 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.
Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation
Publisher: Cold Spring Harbor Laboratory
Date: 17-10-2020
DOI: 10.1101/2020.10.17.343772
Abstract: RNA modifications are dynamic chemical entities that regulate RNA fate, and an avenue for environmental response in neuronal function. However, which RNA modifications may be playing a role in neuronal plasticity and environmental responses 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) isoacceptors 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 play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.
SwiSpot: modeling riboswitches by spotting out switching sequences
Publisher: Oxford University Press (OUP)
Date: 04-07-2016
DOI: 10.1093/BIOINFORMATICS/BTW401
Abstract: 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: www.iet.unipi.it/a.bechini/swispot/ Supplementary information: Supplementary data are available at Bioinformatics online. Contact: a.bechini@ing.unipi.it
Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing
Publisher: Cold Spring Harbor Laboratory
Date: 22-09-2021
DOI: 10.1101/2021.09.22.461331
Abstract: 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 novel 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 any given RNA molecule from its 3’ end, regardless of its polyadenylation status, without the need for PCR lification or ligation of RNA adapters. We demonstrate that Nano3P-seq captures a wide ersity of RNA biotypes, providing quantitative estimates of RNA abundance and tail lengths in mRNA, lncRNA, sn/snoRNA, scaRNA, and rRNA molecules. We find that, in addition to mRNA and lncRNA, polyA tails can be identified in 16S mitochondrial rRNA 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 identify 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 in idual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
Related Organisations
Massachusetts Institute Of Technology
Location: United States of America
Related Funding Activities
Discovery Early Career Researcher Award - Grant ID: DE170100506
Start Date: 02-2017
End Date: 08-2018
Amount: $372,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP180103571
Start Date: 2018
End Date: 08-2019
Amount: $388,950.00
Funder: Australian Research Council
View Funded Activity