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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 N6-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. This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skodowska-Curie grant agreement no. 713673. This work was supported by the Australian Research Council (DP180103571 to E.M.N.) and the MEIC (PGC2018-098152-A-100 to E.M.N.). We acknowledge the support of the MEIC to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya.

Trypanosoma cruzi, the causative agent of Chagas disease, has a complex life cycle that requires the adaptation to different environments. In the absence of traditional mechanisms for regulation of gene expression, this parasite relies on posttranscriptional control events, which allow the progression of its life cycle in different hosts and stress conditions. In this context, different stress conditions trigger the aggregation of RNA-binding proteins and their target mRNAs into cytoplasmic foci known as RNA granules, which act as RNA-sorting centers. In this study, we have characterized the T. cruzi RNA-binding protein ALBA30 during nutritional stress conditions. Using a recombinant form of TcALBA30 to facilitate its detection (rTcALBA30), we showed that this protein resides in the cytoplasm in normal growth conditions but is recruited into cytoplasmic foci after starvation. Moreover, evaluation of rTcALBA30 in parasites that reached the stationary phase of growth also showed the recruitment of this protein into cytoplasmic foci. Our results indicate that, similar to TbALBA3, TcALBA30 aggregates into stress granules in parasites submitted to nutritional stress.

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) paralog of RPL3 (uL3) that is exclusively expressed in muscle and heart tissues, by generating a viable homozygousRpl3lknockout 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.

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m6A), has been implicated in neurogenesis and memory formation. However, whether additional RNA modifications may be playing a role in neuronal functions and in response to environmental queues is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m2,2G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNAAla(AGC) isodecoders at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes may play a role in neuronal plasticity and transmission of information, presumably by addressing new targets. NJ was supported by a UNSW International PhD fellowship. SC is supported by a fellowship from ”la Caixa'' Foundation (LCF/BQ/DI19/11730036). This work was supported by NHMRC funds (Project Grant APP1070631 to JSM), funds from the Australian Research Council (DP180103571 to EMN) and funds from the Garvan Young Investigator Award (to NS). This work was partly supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (PGC2018-098152-A-100 to EMN).The mass spectrometric analyses shown in Figure S3 were performed in the CRG/UPF Proteomics Unit which is part of the of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF

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

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.

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

A broad diversity 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 individually. 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 forde novoprediction 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 individual 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 predictedde novoand in a quantitative manner using native RNA nanopore sequencing.

We characterize a novel human cohesinopathy originated from a familial germline mutation of the gene encoding the cohesin subunit STAG2, which we propose to call STAG2-related X-linked Intellectual Deficiency. Five individuals carry a STAG2 p.Ser327Asn (c.980 G > A) variant that perfectly cosegregates with a phenotype of syndromic mental retardation in a characteristic X-linked recessive pattern. Although patient-derived cells did not show overt sister-chromatid cohesion defects, they exhibited altered cell cycle profiles and gene expression patterns that were consistent with cohesin deficiency. The protein level of STAG2 in patient cells was normal. Interestingly, STAG2 S327 is located at a conserved site crucial for binding to SCC1 and cohesin regulators. When expressed in human cells, the STAG2 p.Ser327Asn mutant is defective in binding to SCC1 and other cohesin subunits and regulators. Thus, decreased amount of intact cohesin likely underlies the phenotypes of STAG2-SXLID. Intriguingly, recombinant STAG2 p.Ser327Asn binds normally to SCC1, WAPL, and SGO1 in vitro, suggesting the existence of unknown in vivo mechanisms that regulate the interaction between STAG2 and SCC1. A newly discovered developmental disease is characterized by mutations in a subunit of the cohesin protein involved in cell division. A team led by Sergio Pena from GENE—Nucleo de Genetica Medica, Brazil, and Hongtao Yu from the University of Texas Southwestern Medical Center, USA, describe a Brazilian family with five male relatives, all with intellectual deficiency, short stature, and other abnormalities. The family tree pointed toward an X-linked pattern of inheritance, so the researchers performed a network analysis of 24 genes on the X chromosome known to contribute to mental retardation. They found that all five individuals had a mutation in a gene called STAG2, which encodes a subunit of cohesin. The mutant STAG2 did not bind properly to other cohesin subunits in human cells, and patient-derived cells exhibited altered cell cycle profiles. The researchers propose calling the disease “STAG2-related X-linked intellectual deficiency”.

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

O Trypanosoma cruzi e considerado um dos eucariotos mais resistentes a Radiação ionizante, suportando doses sub-letais de ate 1,0 kGy. A radiação ionizante do tipo gama promove a quebra das fitas simples e duplas do DNA, além de gerar espécies reativas de oxigênio (ROS). Apos a irradiação, os parasitos cessam o seu crescimento por ate 96 horas, retomando-o em seguida e atingindo a fase estacionaria em ate 240 horas apos irradiação. O padrão de bandas cromossomais, que são extensamente fragmentadas pela radiação, e restabelecido cerca de 48 horas apos o estresse. Para tolerar tais danos, e necessário um eficiente aparato de reconhecimento e reparo de lesões. Neste trabalho, investigamos a radioresistencia em T.cruzi, mediante a análise do seu perfil de expressão protéica e da sua atividade mitocondrial em diferentes tempos apos uma dose de 500 Gy de radiação gama. Utilizando a metodologia 2D-DIGE, foram encontrados 543 spots protéicos diferencialmente expressos apos a exposição do T. cruzi a radiação. A partir destes spots, 53 proteínas foram identificadas por espectrometria de massas (MS/MS) e classificadas de acordo com sua função biológica. Analisando a anotação funcional das 53 proteínas, observamos uma tendência para a superexpressão de proteínas com peso molecular abaixo do predito. Esta tendência indica a possível existência de modificações pos-traducionais e/ou de mecanismos de processamento das proteínas identificadas, que geraria polipeptídios mais curtos do que o predito em resposta a radiação gama. Outras tendências observadas em nossas analises de proteômica incluem alterações nos seguintes processos biológicos: regulação positiva do processo de síntese de proteínas, repressão de proteínas envolvidas no processo de enovelamento (exceto pela indução de duas chaperonas do retículo endoplasmático), repressão das vias de geração de ATP por fosforilação oxidativa, da glicolise, e do metabolismo de aminoácidos. Nossos resultados também indicam que a síntese protéica de#novo e essencial para a recuperação do T. cruzi apos o estresse por exposição a radiação gama, pois o tratamento com inibidores de tradução interfere drasticamente no retorno ao crescimento dos parasitos irradiados. Ao avaliarmos a atividade mitocondrial de T. cruzi, xvi! verificamos que parasitos irradiados produzem mais ATP do que parasitos não irradiados, alem de consumirem mais O2. Adicionalmente, a análise da expressão gênica de T. cruzi irradiado por qRT-PCR mostrou que os genes do maxicírculo mitocondrial foram induzidos apos a irradiação. Ambos os resultados indicam uma maior atividade da mitocôndria apos o estresse. Entretanto, observamos uma menor produção de H2O2 nos parasitos irradiados, indicando assim a existência de um mecanismo eficiente de eliminação de radicais livres. O presente estudo revela a resposta peculiar de T. cruzi a radiação ionizante e suscita questionamentos pertinentes sobre como este organismo e capaz de modificar seu perfil de expressão protéica e sua atividade mitocondrial para possibilitar a sobrevivência em condições tão adversas. The Trypanosoma cruzi is considered as one of the eukaryotes most resistant to ionizing radiation, enduring sub-lethal doses of up to 1.0 kGy. The ionizing radiation by gamma rays promotes double-strand and single-strand breaks on the DNA and, additionally, generates reactive oxygen species (ROS). After irradiation the parasites cease growing for as long as 96 hours, further resuming growth and reaching the stationary phase no later than 240 hours after irradiation. The pattern of chromosomal bands which are extremely fragmented by radiation exposure is restored approximately 48 hours after the stress. To enable the parasite to tolerate such damage condition, an efficient apparatus to recognize and repair lesions is necessary. In the present work we investigate T. cruzi radioresistance by analyzing its protein expression patterns and mitochondrial activity in different time points after a dose of 500 Gy of gamma rays. Using the 2DTDIGE methodology, 543 spots were identified as significantly differentially expressed after T. cruzi exposure to radiation. From these 543 spots, 53 different proteins were identified by mass spectrometry (MS/MS) and classified according to biological function. When the functional annotation of these 53 proteins was analyzed, a tendency of overexpression of proteins with molecular weight lower than predicted was observed. This tendency suggests the presence of post-translational modifications and/or processing mechanisms acting upon the identified proteins, leading to the emergence of peptides which are shorter than predicted in response to gamma radiation exposure. Other tendencies observed on proteomic analysis include alterations on the following biological processes: positive regulation of the protein synthesis process, repression of proteins involved with folding processes (except for two endoplasmic reticulum chaperones, which are induced), repression of ATP generation through oxidative phosphorylation, of glycolysis, and of the amino acids metabolism. Our results also indicate that the de novo protein synthesis is essential for T. cruzi recovery from gamma radiation exposure, since the treatment with translation inhibitors drastically interferes with the growth resumption on irradiated parasites. When the mitochondrial activity of T. cruzi xviii was evaluated, we verified that irradiated parasites generate higher levels of ATP than their non-irradiated counterparts, with an associated higher O2 consumption by the former. Additionally, the gene expression analysis of irradiated T. cruzi using qRT-PCR showed that genes from the mitochondrial maxicircle were induced after radiation exposure. Both of these results point to a higher mitochondrial activity after irradiation-caused stress. Nevertheless, we have also observed a lower H2O2 production by irradiated parasites, thus indicating the existence of an efficient mechanism for free radicals clearance in the organism. The present study unravels the peculiar response of T. cruzi to ionizing radiation and evokes questions about the manner in which this organism is capable of performing drastic changes on its protein expression profile and mitochondrial activity to enable its survival in such adverse conditions.

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.