Clément CARRÉ - Academia.edu (original) (raw)

Papers by Clément CARRÉ

tRNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs) derived from tRNAs. tRFs are... more 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 to study tRFs populations and shed light onto tRNA fragments biogenesis. Indeed, we used small RNAs Illumina sequencing datasets extracted from wild type and mutant Drosophila ovaries affecting two different highly conserved steps of tRNA biogenesis: 5'pre-tRNA processing (RNase-P subunit Rpp30) and tRNA 2'-O-methylation (CG7009 and CG5220). Using our pipeline, we show how defects in tRNA biogenesis affect nuclea...

Genes & Development, 2013

Female-specific repression of male-specific-lethal-2 (msl2) mRNA in Drosophila melanogaster provi... more Female-specific repression of male-specific-lethal-2 (msl2) mRNA in Drosophila melanogaster provides a paradigm for coordinated control of gene expression by RNA-binding complexes. Repression is orchestrated by Sex-lethal (SXL), which binds to the 5′ and 3′ untranslated regions (UTRs) of the mRNA and inhibits splicing in the nucleus and subsequent translation in the cytoplasm. Here we show that SXL ensures msl2 silencing by yet a third mechanism that involves inhibition of nucleocytoplasmic transport of msl2 mRNA. To identify SXL cofactors in msl2 regulation, we devised a two-step purification method termed GRAB (GST pull-down and RNA affinity binding) and identified Held-Out-Wings (HOW) as a component of the msl2 5′ UTR-associated complex. HOW directly interacts with SXL and binds to two sequence elements in the msl2 5′ UTR. Depletion of HOW reduces the capacity of SXL to repress the expression of msl2 reporters without affecting SXL-mediated regulation of splicing or translation. ...

Nucleic Acids Research, 2020

2′-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structu... more 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...

FTSJ1 is a phyllogenetically conserved human 2'-O-methyltransferase (Nm-MTase) which modifies pos... more FTSJ1 is a phyllogenetically conserved human 2'-O-methyltransferase (Nm-MTase) which modifies position 32 as well as the wobble position 34 in the AntiCodon Loop (ACL) of specific tRNAs: tRNA Phe(GAA) , tRNA Trp(CCA) and tRNA Leu(UAA). FTSJ1's loss of function has been linked to Non-Syndromic X-Linked Intellectual Disability (NSXLID), and more recently in cancers. However, the exact molecular mechanisms underlying FTSJ1-related pathogenesis are unknown and a potential extended variety of FTSJ1's tRNA targets hasn't been fully addressed yet. We performed unbiased and comprehensive RiboMethSeq analysis of the Nm profiles for human tRNA population extracted from cells derived from NSXLID patients' blood bearing various characterized loss of function mutations in FTSJ1. In addition, we reported a novel FTSJ1 pathogenic variant from a NSXLID patient bearing a de novo mutation in the FTSJ1 gene. Some of the newly identified FTSJ1's tRNA targets are also conserved in Drosophila as shown by our previous study on the fly homologues Trm7_32 and Trm7_34, whose loss affects small RNA silencing pathways. In the current study, we reveal a conserved deregulation in both the miRNA and mRNA populations when FTSJ1 function is compromised. In addition, a cross-analysing between deregulated miRNA and mRNA obtained in FTSJ1 mutants highlighted upregulation of miR10a-5p which has the capacity to silence the SPARC gene mRNA, downregulated in FTSJ1 mutant cells. This suggests that FTSJ1 loss may influence gene expression deregulation by modulation of miRNA silencing. A gene-ontology (GO) enrichment analysis of the deregulated mRNAs primarily matched to brain morphogenesis terms, followed by metabolism and translation related genes. In parallel, the deregulated miRNAs are mostly known for their implication in brain functions and cancers. Based on these results, we suggest that miRNA silencing variations may play a role in the pathological mechanisms of FTSJ1-dependent NSXLID. Finally, our results highlight miR-181a-5p as a potential companion diagnostic test in clinical settings for FTSJ1-related intellectual disability. .

FTSJ1 is a conserved human 2’-O-methyltransferase (Nm-MTase) that modifies several transfer RNAs ... more FTSJ1 is a conserved human 2’-O-methyltransferase (Nm-MTase) that modifies several transfer RNAs (tRNAs) at position 32 and the wobble position 34 in the AntiCodon Loop (ACL). Its loss of function has been linked to Non-Syndromic X-Linked Intellectual Disability (NSXLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here we report a novel FTSJ1 pathogenic variant from a NSXLID patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation (Nm) on all tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA...

2-O-methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structur... more 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 novel Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1, respectively. 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 subsequently, CG5220 methylates position C32 in the same tRNAs and targets also additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as life span reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characteriza...

Genes, 2019

Nm (2’-O-methylation) is one of the most common modifications in the RNA world. It has the potent... more Nm (2’-O-methylation) is one of the most common modifications in the RNA world. It has the potential to influence the RNA molecules in multiple ways, such as structure, stability, and interactions, and to play a role in various cellular processes from epigenetic gene regulation, through translation to self versus non-self recognition. Yet, building scientific knowledge on the Nm matter has been hampered for a long time by the challenges in detecting and mapping this modification. Today, with the latest advancements in the area, more and more Nm sites are discovered on RNAs (tRNA, rRNA, mRNA, and small non-coding RNA) and linked to normal or pathological conditions. This review aims to synthesize the Nm-associated human diseases known to date and to tackle potential indirect links to some other biological defects.

Non-coding RNA Investigation, 2018

Frontiers in Bioengineering and Biotechnology, 2018

Analogous to DNA methylation and histone modifications, RNA modifications represent a novel layer... more Analogous to DNA methylation and histone modifications, RNA modifications represent a novel layer of regulation of gene expression. The dynamic nature and increasing number of RNA modifications offer new possibilities to rapidly alter gene expression upon specific environmental changes. Recent lines of evidence indicate that modified RNA molecules and associated complexes regulating and "reading" RNA modifications play key roles in the nervous system of several organisms, controlling both, its development and function. Mutations in several human genes that modify transfer RNA (tRNA) have been linked to neurological disorders, in particular to intellectual disability. Loss of RNA modifications alters the stability of tRNA, resulting in reduced translation efficiency and generation of tRNA fragments, which can interfere with neuronal functions. Modifications present on messenger RNAs (mRNAs) also play important roles during brain development. They contribute to neuronal growth and regeneration as well as to the local regulation of synaptic functions. Hence, potential combinatorial effects of RNA modifications on different classes of RNA may represent a novel code to dynamically fine tune gene expression during brain function. Here we discuss the recent findings demonstrating the impact of modified RNAs on neuronal processes and disorders.

RNA (New York, N.Y.), Dec 14, 2018

piRNA-mediated repression of transposable elements (TE) in the germline limits the accumulation o... more piRNA-mediated repression of transposable elements (TE) in the germline limits the accumulation of heritable mutations caused by their transposition in the genome. It is not clear whether the piRNA pathway plays a functional role in adult, non-gonadal tissues in Drosophila melanogaster. To address this question, we first analyzed the small RNA content of adult Drosophila melanogaster heads. We found that varying amount of piRNA-sized, ping-pong positive molecules in heads correlates with contamination by gonadal tissue during RNA extraction, suggesting that most of piRNAs detected in head sequencing libraries 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 affect significantly these 24-28 RNA molecules. Instead, we observe incr...

RNA biology, Jan 9, 2018

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA.... more The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found i...

Cell reports, Jan 13, 2018

The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as ... more The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5) RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover...

Non-Coding RNA, 2015

The number of papers dealing with new modus operandi or new biological functions of non-coding RN... more The number of papers dealing with new modus operandi or new biological functions of non-coding RNAs published in recent years has indeed exploded. A simple search for ‘non-coding RNA’ in Pubmed on 10 June 2015 yielded 128,649 articles, half of which were published in the last 10 years [1]. Every researcher in this field knows that he has something to learn and can discover new ideas, new concepts or new tools from studies made in models others than the ones used in its lab. The Scientific board of Non-Coding RNA publishes here its first Journal Club and highlights, in about hundred words, a selection of the most interesting papers published recently. We hope we will tease your curiosity and encourage you to read full papers outside of your research area that you may not have read otherwise. [...]

Methods in molecular biology (Clifton, N.J.), 2014

The tombusvirus P19 VSR (viral suppressor of RNA interference) binds siRNAs with high affinity, w... more The tombusvirus P19 VSR (viral suppressor of RNA interference) binds siRNAs with high affinity, whereas the Flockhouse Virus (FHV) B2 VSR binds both long double-stranded RNA (dsRNA) and small interfering RNAs (siRNAs). Both VSRs are small proteins and function in plant and animal cells. Fusing a Nuclear Localization Signal (NLS) to the N-terminus shifts the localization of the VSR from cytoplasmic to nuclear, allowing researchers to specifically probe the subcellular distribution of siRNAs, and to investigate the function of nuclear and cytoplasmic siRNAs. This chapter provides a detailed protocol for the immunoprecipitation of siRNAs bound to epitope-tagged VSR and subsequent analysis by 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-end-labeling using cytidine-3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-bis phosphate ([5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-(32)P]pCp) and northern blotting.

Science, 2005

All animals coordinate growth and maturation to reach their final size and shape. In insects, ins... more All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. We show that 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP . We also demonstrate that the larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size.

RNA, 2003

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the... more RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21–23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of o...

PLoS ONE, 2013

A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic tran... more A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided.

Molecular and Cellular Biology, 2011

Small GTPases share a biochemical mechanism and act as binary molecular switches. One important f... more Small GTPases share a biochemical mechanism and act as binary molecular switches. One important function of small GTPases in the cell is nucleocytoplasmic transport of both proteins and RNA. Here, we show the stable association of human GPN1 and GPN3, small GTPases related to Ran, with RNA polymerase II (RNAPII) isolated from either the cytoplasmic or nuclear fraction. GPN1 and GPN3 directly interact with RNAPII subunit 7 (RPB7)/RPB4 and the C-terminal domain (CTD) of RNAPII. Depletion of GPN1 or GPN3 using small interfering RNAs led to decreased RNAPII levels in the nucleus and an accumulation of this enzyme in the cytoplasm of human cells. Furthermore, isolation of a GPN1/GPN3/RNAPII complex from stable cell lines expressing a dominant negative GPN1 harboring mutations in the GTP-binding pocket demonstrated a role for these proteins in nuclear import of RNAPII. Thus, GPN1/GPN3 define a new family of small GTPases that are specialized for the transport of RNA polymerase II into the...

Development, 2005

Loss of function of either the ecdysone receptor (EcR) or Ultraspiracle(USP), the two components ... more Loss of function of either the ecdysone receptor (EcR) or Ultraspiracle(USP), the two components of the ecdysone receptor, causes precocious differentiation of the sensory neurons on the wing of Drosophila. We propose that the unliganded receptor complex is repressive and that this repression is relieved as the hormone titers increase at the onset of metamorphosis. The point in development where the receptor complex exerts this repression varies for different groups of sensilla. For the chemosensory organ precursors along the wing margin, the block is at the level of senseless expression and is indirect, via the repressive control of broad expression. Misexpressing broad or senselesscan circumvent the repression by the unliganded receptor and leads to precocious differentiation of the sensory neurons. This precocious differentiation results in the misguidance of their axons. The sensory precursors of some of the campaniform sensilla on the third longitudinal vein are born prior to t...