Smurfness-based two-phase model of ageing helps deconvolve the ageing transcriptional signature.

Ageing is characterised at the molecular level by six transcriptional 'hallmarks of ageing', that are commonly described as progressively affected as time passes. By contrast, the 'Smurf' assay separates high-and-constant-mortality risk individuals from healthy, zero-mortality risk individuals, based on increased intestinal permeability. Performing whole body total RNA sequencing, we found that Smurfness distinguishes transcriptional changes associated with chronological age from those associated with biological age. We show that transcriptional heterogeneity increases with chronological age in non-Smurf individuals preceding the other five hallmarks of ageing that are specifically associated with the Smurf state. Using this approach, we also devise targeted pro-longevity genetic interventions delaying entry in the Smurf state. We anticipate that increased attention to the evolutionary conserved Smurf phenotype will bring about significant advances in our understanding of the mechanisms of ageing.

Cellular senescence in malignant cells promotes tumor progression in mouse and patient Glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, yet it remains refractory to systemic therapy. Elimination of senescent cells has emerged as a promising new treatment approach against cancer. Here, we investigated the contribution of senescent cells to GBM progression. Senescent cells are identified in patient and mouse GBMs. Partial removal of p16Ink4a-expressing malignant senescent cells, which make up less than 7 % of the tumor, modifies the tumor ecosystem and improves the survival of GBM-bearing female mice. By combining single cell and bulk RNA sequencing, immunohistochemistry and genetic knockdowns, we identify the NRF2 transcription factor as a determinant of the senescent phenotype. Remarkably, our mouse senescent transcriptional signature and underlying mechanisms of senescence are conserved in patient GBMs, in whom higher senescence scores correlate with shorter survival times. These findings suggest that senolytic drug therapy may be a beneficial adjuvant therapy for patients with GBM.

Glioblastoma cell motility depends on enhanced oxidative stress coupled with mobilization of a sulfurtransferase.

Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients' glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients' tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients' tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients' tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.

Cytoplasmic forces functionally reorganize nuclear condensates in oocytes.

Cells remodel their cytoplasm with force-generating cytoskeletal motors. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic and germ cells. These forces are transmitted inside the nucleus, yet their consequences on liquid-like biomolecular condensates residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include nuclear speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of nuclear condensates for the success of meiotic divisions. These cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Disrupting the forces decelerates nuclear condensate reorganization on both scales, which correlates with compromised condensate-associated mRNA processing and hindered oocyte divisions that drive female fertility. We establish that cytoplasmic forces can reorganize nuclear condensates in an evolutionary conserved fashion in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections.

tRNA Fragments Populations Analysis in Mutants Affecting tRNAs Processing and tRNA Methylation.

tRNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs) derived from tRNAs. tRFs are highly abundant in many cell types including stem cells and cancer cells, and are found in all domains of life. Beyond translation control, tRFs have several functions ranging from transposon silencing to cell proliferation control. However, the analysis of tRFs presents specific challenges and their biogenesis is not well understood. They are very heterogeneous and highly modified by numerous post-transcriptional modifications. Here we describe a bioinformatic pipeline (tRFs-Galaxy) to study tRFs populations and shed light onto tRNA fragments biogenesis in Drosophila melanogaster. Indeed, we used small RNAs Illumina sequencing datasets extracted from wild type and mutant ovaries affecting two different highly conserved steps of tRNA biogenesis: 5'pre-tRNA processing (RNase-P subunit Rpp30) and tRNA 2'-O-methylation (dTrm7_34 and dTrm7_32). Using our pipeline, we show how defects in tRNA biogenesis affect nuclear and mitochondrial tRFs populations and other small non-coding RNAs biogenesis, such as small nucleolar RNAs (snoRNAs). This tRF analysis workflow will advance the current understanding of tRFs biogenesis, which is crucial to better comprehend tRFs roles and their implication in human pathology.

A single-cell RNA-sequencing training and analysis suite using the Galaxy framework.

BACKGROUND: The vast ecosystem of single-cell RNA-sequencing tools has until recently been plagued by an excess of diverging analysis strategies, inconsistent file formats, and compatibility issues between different software suites. The uptake of 10x Genomics datasets has begun to calm this diversity, and the bioinformatics community leans once more towards the large computing requirements and the statistically driven methods needed to process and understand these ever-growing datasets. RESULTS: Here we outline several Galaxy workflows and learning resources for single-cell RNA-sequencing, with the aim of providing a comprehensive analysis environment paired with a thorough user learning experience that bridges the knowledge gap between the computational methods and the underlying cell biology. The Galaxy reproducible bioinformatics framework provides tools, workflows, and trainings that not only enable users to perform 1-click 10x preprocessing but also empower them to demultiplex raw sequencing from custom tagged and full-length sequencing protocols. The downstream analysis supports a range of high-quality interoperable suites separated into common stages of analysis: inspection, filtering, normalization, confounder removal, and clustering. The teaching resources cover concepts from computer science to cell biology. Access to all resources is provided at the singlecell.usegalaxy.eu portal. CONCLUSIONS: The reproducible and training-oriented Galaxy framework provides a sustainable high-performance computing environment for users to run flexible analyses on both 10x and alternative platforms. The tutorials from the Galaxy Training Network along with the frequent training workshops hosted by the Galaxy community provide a means for users to learn, publish, and teach single-cell RNA-sequencing analysis.

tRNA 2'-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila.

2'-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

GC content shapes mRNA storage and decay in human cells.

mRNA translation and decay appear often intimately linked although the rules of this interplay are poorly understood. In this study, we combined our recent P-body transcriptome with transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors, and with available CLIP and related data. This revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA stability: P-bodies contain mostly AU-rich mRNAs, which have a particular codon usage associated with a low protein yield; AU-rich and GC-rich transcripts tend to follow distinct decay pathways; and the targets of sequence-specific RBPs and miRNAs are also biased in terms of GC content. Altogether, these results suggest an integrated view of post-transcriptional control in human cells where most translation regulation is dedicated to inefficiently translated AU-rich mRNAs, whereas control at the level of 5' decay applies to optimally translated GC-rich mRNAs.

Capture at the single cell level of metabolic modules distinguishing aggressive and indolent glioblastoma cells.

Glioblastoma cell ability to adapt their functioning to microenvironment changes is a source of the extensive intra-tumor heterogeneity characteristic of this devastating malignant brain tumor. A systemic view of the metabolic pathways underlying glioblastoma cell functioning states is lacking. We analyzed public single cell RNA-sequencing data from glioblastoma surgical resections, which offer the closest available view of tumor cell heterogeneity as encountered at the time of patients' diagnosis. Unsupervised analyses revealed that information dispersed throughout the cell transcript repertoires encoded the identity of each tumor and masked information related to cell functioning states. Data reduction based on an experimentally-defined signature of transcription factors overcame this hurdle. It allowed cell grouping according to their tumorigenic potential, regardless of their tumor of origin. The approach relevance was validated using independent datasets of glioblastoma cell and tissue transcriptomes, patient-derived cell lines and orthotopic xenografts. Overexpression of genes coding for amino acid and lipid metabolism enzymes involved in anti-oxidative, energetic and cell membrane processes characterized cells with high tumorigenic potential. Modeling of their expression network highlighted the very long chain polyunsaturated fatty acid synthesis pathway at the core of the network. Expression of its most downstream enzymatic component, ELOVL2, was associated with worsened patient survival, and required for cell tumorigenic properties in vivo. Our results demonstrate the power of signature-driven analyses of single cell transcriptomes to obtain an integrated view of metabolic pathways at play within the heterogeneous cell landscape of patient tumors.

Dual-layer transposon repression in heads of Drosophila melanogaster.

piRNA-mediated repression of transposable elements (TE) in the germline limits the accumulation of mutations caused by their transposition. It is not clear whether the piRNA pathway plays a role in adult, nongonadal tissues in Drosophila melanogaster. To address this question, we analyzed the small RNA content of adult Drosophila melanogaster heads. We found that the varying amount of piRNA-sized, ping-pong positive molecules in heads correlates with contamination by gonadal tissue during RNA extraction, suggesting that most of the piRNAs detected in heads originate from gonads. We next sequenced the heads of wild-type and piwi mutants to address whether piwi loss of function would affect the low amount of piRNA-sized, ping-pong negative molecules that are still detected in heads hand-checked to avoid gonadal contamination. We find that loss of piwi does not significantly affect these 24-28 nt RNAs. Instead, we observe increased siRNA levels against the majority of Drosophila TE families. To determine the effect of this siRNA level change on transposon expression, we sequenced the transcriptome of wild-type, piwi, dicer-2 and piwi, dicer-2 double-mutant heads. We find that RNA expression levels of the majority of TE in piwi or dicer-2 mutants remain unchanged and that TE transcripts increase only in piwi, dicer-2 double-mutants. These results lead us to suggest a dual-layer model for TE repression in adult somatic tissues. Piwi-mediated gene silencing established during embryogenesis constitutes the first layer of TE repression whereas Dicer-2-dependent siRNA-mediated silencing provides a backup mechanism to repress TEs that escape silencing by Piwi.

Small-RNA sequencing identifies dynamic microRNA deregulation during skeletal muscle lineage progression.

Skeletal muscle satellite cells are quiescent adult resident stem cells that activate, proliferate and differentiate to generate myofibres following injury. They harbour a robust proliferation potential and self-renewing capacity enabling lifelong muscle regeneration. Although several classes of microRNAs were shown to regulate adult myogenesis, systematic examination of stage-specific microRNAs during lineage progression from the quiescent state is lacking. Here we provide a genome-wide assessment of the expression of small RNAs during the quiescence/activation transition and differentiation by RNA-sequencing. We show that the majority of small RNAs present in quiescent, activated and differentiated muscle cells belong to the microRNA class. Furthermore, by comparing expression in distinct cell states, we report a massive and dynamic regulation of microRNAs, both in numbers and amplitude, highlighting their pivotal role in regulation of quiescence, activation and differentiation. We also identify a number of microRNAs with reliable and specific expression in quiescence including several maternally-expressed miRNAs generated at the imprinted Dlk1-Dio3 locus. Unexpectedly, the majority of class-switching miRNAs are associated with the quiescence/activation transition suggesting a poised program that is actively repressed. These data constitute a key resource for functional analyses of miRNAs in skeletal myogenesis, and more broadly, in the regulation of stem cell self-renewal and tissue homeostasis.

Metavisitor, a Suite of Galaxy Tools for Simple and Rapid Detection and Discovery of Viruses in Deep Sequence Data.

Metavisitor is a software package that allows biologists and clinicians without specialized bioinformatics expertise to detect and assemble viral genomes from deep sequence datasets. The package is composed of a set of modular bioinformatic tools and workflows that are implemented in the Galaxy framework. Using the graphical Galaxy workflow editor, users with minimal computational skills can use existing Metavisitor workflows or adapt them to suit specific needs by adding or modifying analysis modules. Metavisitor works with DNA, RNA or small RNA sequencing data over a range of read lengths and can use a combination of de novo and guided approaches to assemble genomes from sequencing reads. We show that the software has the potential for quick diagnosis as well as discovery of viruses from a vast array of organisms. Importantly, we provide here executable Metavisitor use cases, which increase the accessibility and transparency of the software, ultimately enabling biologists or clinicians to focus on biological or medical questions.

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes.

Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O'nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.

Paramutation in Drosophila Requires Both Nuclear and Cytoplasmic Actors of the piRNA Pathway and Induces Cis-spreading of piRNA Production.

Transposable element activity is repressed in the germline in animals by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. We have shown that, in Drosophila melanogaster, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. We report here that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff, and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21-nt RNAs, which are produced by the paramutated cluster, in addition to 23- to 28-nt piRNAs are not necessary for paramutation to occur. Production of these 21-nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus that produces piRNAs along the whole length of the transgenes. Finally, we observed that maternally inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between nonfully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.

tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription.

RNase P is a conserved endonuclease that processes the 5' trailer of tRNA precursors. We have isolated mutations in Rpp30, a subunit of RNase P, and find that these induce complete sterility in Drosophila females. Here, we show that sterility is not due to a shortage of mature tRNAs, but that atrophied ovaries result from the activation of several DNA damage checkpoint proteins, including p53, Claspin, and Chk2. Indeed, we find that tRNA processing defects lead to increased replication stress and de-repression of transposable elements in mutant ovaries. We also report that transcription of major piRNA sources collapse in mutant germ cells and that this correlates with a decrease in heterochromatic H3K9me3 marks on the corresponding piRNA-producing loci. Our data thus link tRNA processing, DNA replication, and genome defense by small RNAs. This unexpected connection reveals constraints that could shape genome organization during evolution.

The cricket paralysis virus suppressor inhibits microRNA silencing mediated by the Drosophila Argonaute-2 protein.

Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.

bacterial infection drives the expression dynamics of microRNAs and their isomiRs.

The optimal coordination of the transcriptional response of host cells to infection is essential for establishing appropriate immunological outcomes. In this context, the role of microRNAs (miRNAs)--important epigenetic regulators of gene expression--in regulating mammalian immune systems is increasingly well recognised. However, the expression dynamics of miRNAs, and that of their isoforms, in response to infection remains largely unexplored. Here, we characterized the genome-wide miRNA transcriptional responses of human dendritic cells, over time, to various mycobacteria differing in their virulence as well as to other bacteria outside the genus Mycobacterium, using small RNA-sequencing. We detected the presence of a core temporal response to infection, shared across bacteria, comprising 49 miRNAs, highlighting a set of miRNAs that may play an essential role in the regulation of basic cellular responses to stress. Despite such broadly shared expression dynamics, we identified specific elements of variation in the miRNA response to infection across bacteria, including a virulence-dependent induction of the miR-132/212 family in response to mycobacterial infections. We also found that infection has a strong impact on both the relative abundance of the miRNA hairpin arms and the expression dynamics of miRNA isoforms. That we observed broadly consistent changes in relative arm expression and isomiR distribution across bacteria suggests that this additional, internal layer of variability in miRNA responses represents an additional source of subtle miRNA-mediated regulation upon infection. Collectively, this study increases our understanding of the dynamism and role of miRNAs in response to bacterial infection, revealing novel features of their internal variability and identifying candidate miRNAs that may contribute to differences in the pathogenicity of mycobacterial infections.

Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.

Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o'nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens.

Computing siRNA and piRNA overlap signatures.

High-throughput sequencing approaches opened the possibility to precisely map full populations of small RNAs to the genomic loci from which they originate. A bioinformatic approach revealed a strong tendency of sense and antisense piRNAs to overlap with each other over ten nucleotides and had a major role in understanding the mechanisms of piRNA biogenesis. Using similar approaches, it is possible to detect a tendency of sense and antisense siRNAs to overlap over 19 nucleotides. Thus, the so-called overlap signature which describes the tendency of small RNA to map in a specific way relative to each other has become the approach of choice to identify and characterize specific classes of small RNAs. Although simple in essence, the bioinformatic methods used for this approach are not easily accessible to biologists. Here we provide a python software that can be run on most of desktop or laptop computers to compute small RNA signatures from files of sequencing read alignments. Moreover, we describe and illustrate step by step two different algorithms at the core of the software and which were previously used in a number of works.