168 research outputs found
SwiSpot: Modeling riboswitches by spotting out switching sequences
No full textMotivation: 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
EpiNano: detection of m6A RNA modifications using Oxford nanopore direct RNA sequencing
No full textRNA 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 m6A RNA modifications from dRNA-seq datasets. The latest version of EpiNano contains models for predicting m6A 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 m6A RNA modifications in dRNA-seq data.We thank all members of the Novoa lab for their valuable insights and discussion. We thank Rebeca Medina for obtaining the TapeStation image used for Fig. 1. OB is supported by an international PhD fellowship (UIPA) from the University of New South Wales. This work was supported by the Australian Research Council (DP180103571 to EMN) and the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (PGC2018-098152-A-100 to EMN). We acknowledge the support of the MEIC to the EMBL partnership, Centro de Excelencia Severo Ochoa, and CERCA Program/Generalitat de Catalunya
Investigating sperm RNA dynamics in intergenerational inheritance using nanopore sequencing
No full textThe aim of this thesis is to delve into sperm RNA dynamics during maturation and paternal inheritance. To obtain an integrative multi-omics view of sperm RNA during these processes, I turned to the new direct RNA sequencing platform, nanopore sequencing, from Oxford Nanopore Technologies. This technology has the potential to capture information about the transcriptome, epitranscriptome, degradation patterns, and 3’ end and tail composition, in the full compendium of RNA biotypes, within a single sequencing run. The first half of this thesis is devoted to developing RNA modification detection and short RNA sequencing methodologies for nanopore sequencing. In the second half of this thesis, I apply these methodologies to characterise sperm RNA dynamics during maturation and upon high-fat diet perturbation. I also investigate the effect of high-fat diet timing on the intergenerational inheritance of acquired metabolic disorders with the view of identifying key intervention points.L'objectiu d'aquesta tesi era aprofundir en les dinàmiques de l'ARN espermàtic durant la maduració i l'herència paterna. Per obtenir una visió complerta i integradora de l'ARN espermàtic durant aquests processos, vaig recórrer a la nova plataforma de seqüenciació directa d'ARN, la seqüenciació mitjançant el ús de nanopors, una tècnica desenvolupada per Oxford Nanopore Technologies. Aquesta tecnologia té el potencial de capturar informació sobre el transcriptoma, l'epitranscriptoma, els patrons de degradació, la composició de l'extrem 3' i la cua poliadelinada i; detectar tots els biotips d'ARN a la vegada en una únic experiment. La primera meitat d'aquesta tesi es dedica a desenvolupar metodologies de detecció de modificacions d'ARN i seqüenciació curta d'ARN per a ser aplicades a la seqüenciació de nanopors. En la segona meitat, aplico aquestes metodologies per caracteritzar les dinàmiques de l'ARN espermàtic durant la maduració i les induides per una dieta amb un alt contingut de greixos. També investigo l'efecte d’aquest tipus de dieta en l'herència intergeneracional dels trastorns metabòlics adquirits amb l'objectiu d'identificar els punts clau d'intervenció.Programa de doctorat en Biomedicin
Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo
No full textMalaria 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.Human Frontier Science Program (Strasbourg, France) (Postdoctoral Fellowship LT000307/2013
Highly evolvable malaria vectors: The genomes of 16 Anopheles mosquitoes
No full textVariation 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.National Human Genome Research Institute (U.S.) (U54 HG003067
Evolution of the gene translation machinery and its applications to drug discovery
No full text[eng] Gene translation is a central process in all cells, in which messenger RNA (mRNA) is decoded by the ribosome to produce a specific amino acid chain, that will later fold into an active protein. This process is facilitated by transfer RNAs (tRNAs), which carry the specific amino acids, and bind with its complementary anticodon sequences to that of the mRNA. The correct charging of the tRNA is catalyzed by aminoacyl-tRNA synthetases, and thus, are responsible for stablishing the genetic code.
Despite the central role of tRNAs in protein translation, the connections between tRNA gene population dynamics and genome evolution have rarely been explored. In this work we have characterized the evolution of genomes through the study of its tRNA populations. We find that its evolution is linked to the appearance of diverse strategies to maximize translation efficiency. Indeed, these diverse strategies rise due to the appearance of two tRNA modification enzymes, which cause a selective enrichment of specific tRNA isoacceptors, and consequently, the phenomenon of codon usage bias.
Furthermore, we have characterized with greater detail the gene translation machinery of Plasmodium falciparum, the most deadly form of the Plasmodium genus causing malaria. To decipher novel compounds that inhibit parasite growth, we have designed and tested several drug design strategies both in slico and in vitro, finding some promising molecules that kill the parasite without damaging human cells, and that show in vivo activity against P.yoelii-infected mice.[spa] La traducción de proteinas es un proceso central en todas las células, en el cual el ARN mensajero es descodificado en el ribosoma para producir una cadena aminoacídica, que después se plegará dando lugar a una proteina activa. Este proceso está facilitado por los ARN de transferencia (ARNt), que llevan unidos covalentemente amino ácidos específicos. La unión precisa de cada amino ácido a su ARNt está catalizada por las aminoacil-ARNt sintetasas, y por tanto, estas enzimas son las responsables de establecimiento del código genético.
A pesar del papel central de los ARNt en la traducción de proteinas, las conexiones entre la dinámica de la población de genes de ARNt y su evolución a través de los distintos genomas no se ha estudiado. En este trabajo hemos caracterizado la evolución de las especies desde el estudio de sus poblaciones de ARNt, encontrando que su evolución está ligada a la aparición de distintas estrategias de maximización de la eficiencia de traducción. A su vez, estas distintas estrategias surgen por la aparición de distinas enzimas de modificación del ARNt en diversos puntos de la evolución, causando en gran medida el fenómeno del uso desigual de codones entre las distinas especies, y entre los genes dentro de una misma especie.
Además, en este trabajo se ha caracterizado con mayor detalle la maquinaria de traducción de Plasmodium falciparum, la especie causante del mayor número de muertes anuales por malaria. Para intentar encontrar fármacos que inhiban al parásito, hemos diseñado distintas estrategias de diseño de fármacos, y hemos testeado varias librerias contra eritrocitos infectados por este parásito, encontrando algunas prometedoras moleculas que inhiben al parásito sin causar citotoxicidad en células humanas, y que funcionan también funcionan in vivo en modelos de ratón
Flaix de ciència. Seguint les empremtes de la traducció genètica. El paper de les modificacions dels RNA de transferència en l’evolució dels genomes Premi SCB al Jove Investigador
No full textThird-generation sequencing: a journey to the world of RNA modifications (Seminaris Tecnològics 2020)
No full textHuman tRNAs with inosine 34 are essential to efficiently translate eukarya-specific low-complexity proteins
No full textThe modification of adenosine to inosine at the wobble position (I34) of tRNA anticodons is an abundant and essential feature of eukaryotic tRNAs. The expansion of inosine-containing tRNAs in eukaryotes followed the transformation of the homodimeric bacterial enzyme TadA, which generates I34 in tRNAArg and tRNALeu, into the heterodimeric eukaryotic enzyme ADAT, which modifies up to eight different tRNAs. The emergence of ADAT and its larger set of substrates, strongly influenced the tRNA composition and codon usage of eukaryotic genomes. However, the selective advantages that drove the expansion of I34-tRNAs remain unknown. Here we investigate the functional relevance of I34-tRNAs in human cells and show that a full complement of these tRNAs is necessary for the translation of low-complexity protein domains enriched in amino acids cognate for I34-tRNAs. The coding sequences for these domains require codons translated by I34-tRNAs, in detriment of synonymous codons that use other tRNAs. I34-tRNA-dependent low-complexity proteins are enriched in functional categories related to cell adhesion, and depletion in I34-tRNAs leads to cellular phenotypes consistent with these roles. We show that the distribution of these low-complexity proteins mirrors the distribution of I34-tRNAs in the phylogenetic tree.Spanish Ministry of Economy and Competitiveness [PID2019-108037RB-100 to L.R.d.P., PGC2018-099921 to T.G., PGC2018-098152-A-100 to E.M.N]; Australian Research Council [DP180103571 to E.M.N]; European Union's Horizon 2020 research and innovation programme [ERC-2016–724173 to T.G.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness.Peer Reviewed"Article signat per 14 autors/es: Adrian Gabriel Torres, Marta Rodríguez-Escribà, Marina Marcet-Houben, Helaine Graziele Santos Vieira, Noelia Camacho, Helena Catena, Marina Murillo Recio, Àlbert Rafels-Ybern, Oscar Reina, Francisco Miguel Torres, Ana Pardo-Saganta, Toni Gabaldón, Eva Maria Novoa, Lluís Ribas de Pouplana"Postprint (author's final draft
Decoding the epitranscriptomic complexity through computational methods based on long-read sequencing
No full textRNA modifications, collectively known as the ‘epitranscriptome’, play a major role in multiple
biological processes, including splicing regulation, gene expression regulation and antibiotic
resistance. Unfortunately, next-generation sequencing (NGS) based methods rely on reverse
transcription and sequencing-by-synthesis processes that are blind to modified bases. Here,
we propose to develop several computational approaches that allow the detection of RNA
modifications at position and/or single molecule level using datasets obtained from direct RNA
sequencing (DRS), a platform developed by Oxford Nanopore Technologies (ONT). Then, we
aim to employ these methods to study the role of these modifications in: i) antibiotic resistance
mechanisms mediated by ribosomal RNA modifications; ii) the role of RNA modifications in
single-stranded RNA viruses, and iii) vertebrate embryogenesis, specifically in the maternal-
to-zygotic transition (MZT) of zebrafish.El conjunt de les modificacions de l’ARN, conegut com a epitranscriptoma, té un paper
relevant en molts processos biologics, com ara la regulació de l’expressió gènica, el splicing
alternatiu i en mecanismes de resitencia antibiòtica. Malauradament, els mètodes per detectar
aquestes modificacions estan basades en la transcripció inversa i en la seqüenciació per
síntesis i per tant, no poden detectar directament la presència d’aquestes bases modificades.
Durant aquesta tesis, es desenvolupen diversos mètodes computacionals per identificar
aquestes modificacions amb diferents resolucions utilitzant dades generades per direct RNA
sequencing (DRS), una tècnica de seqüenciació desenvolupada per Oxford Nanopore
Technologies (ONT). Després, aquests mètodes s’utilitzen per estudiar el paper d’aquestes
modificacions en diferents contextos biologics: i) mecanismes de resistència antibiòtica
mediats per les modificacions de l’ARN ribosomal; ii) en virus d’ARN de cadena única i; iii) en
la embriogènesis en vertebrats, concretament durant la maternal-to-zygotic transition (MZT)
en el peix zebra.Programa de Doctorat en Biomedicin
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