Publications by authors named "Antoine Graindorge"

10 Publications

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Author Correction: In-cell identification and measurement of RNA-protein interactions.

Nat Commun 2020 07 8;11(1):3498. Epub 2020 Jul 8.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, 75005, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17282-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343817PMC
July 2020

In-cell identification and measurement of RNA-protein interactions.

Nat Commun 2019 11 22;10(1):5317. Epub 2019 Nov 22.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, 75005, France.

Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.
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http://dx.doi.org/10.1038/s41467-019-13235-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876571PMC
November 2019

Strategies for genetic inactivation of long noncoding RNAs in zebrafish.

RNA 2019 08 1;25(8):897-904. Epub 2019 May 1.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, 75005 Paris, France.

The number of annotated long noncoding RNAs (lncRNAs) continues to grow; however, their functional characterization in model organisms has been hampered by the lack of reliable genetic inactivation strategies. While partial or full deletions of lncRNA loci disrupt lncRNA expression, they do not permit the formal association of a phenotype with the encoded transcript. Here, we examined several alternative strategies for generating lncRNA null alleles in zebrafish and found that they often resulted in unpredicted changes to lncRNA expression. Removal of the transcription start sites (TSSs) of lncRNA genes resulted in hypomorphic mutants, due to the usage of either constitutive or tissue-specific alternative TSSs. Deletions of short, highly conserved lncRNA regions can also lead to overexpression of truncated transcripts. In contrast, knock-in of a polyadenylation signal enabled complete inactivation of , the most abundant vertebrate lncRNA. In summary, lncRNA null alleles require extensive in vivo validation, and we propose insertion of transcription termination sequences as the most reliable approach to generate lncRNA-deficient zebrafish.
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http://dx.doi.org/10.1261/rna.069484.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633201PMC
August 2019

Hrp48 and eIF3d contribute to msl-2 mRNA translational repression.

Nucleic Acids Res 2018 05;46(8):4099-4113

Gene Regulation, Stem Cells and Cancer Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 08003 Barcelona, Spain.

Translational repression of msl-2 mRNA in females of Drosophila melanogaster is an essential step in the regulation of X-chromosome dosage compensation. Repression is orchestrated by Sex-lethal (SXL), which binds to both untranslated regions (UTRs) of msl-2 and inhibits translation initiation by poorly understood mechanisms. Here we identify Hrp48 as a SXL co-factor. Hrp48 binds to the 3' UTR of msl-2 and is required for optimal repression by SXL. Hrp48 interacts with eIF3d, a subunit of the eIF3 translation initiation complex. Reporter and RNA chromatography assays showed that eIF3d binds to msl-2 5' UTR, and is required for efficient translation and translational repression of msl-2 mRNA. In line with these results, eIF3d depletion -but not depletion of other eIF3 subunits- de-represses msl-2 expression in female flies. These data are consistent with a model where Hrp48 inhibits msl-2 translation by targeting eIF3d. Our results uncover an important step in the mechanism of msl-2 translation regulation, and illustrate how general translation initiation factors can be co-opted by RNA binding proteins to achieve mRNA-specific control.
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http://dx.doi.org/10.1093/nar/gky246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934621PMC
May 2018

Sex-lethal promotes nuclear retention of msl2 mRNA via interactions with the STAR protein HOW.

Genes Dev 2013 Jun;27(12):1421-33

Gene Regulation, Stem Cells, and Cancer Programme, Centre for Genomic Regulation (CRG), 08003 Barcelona, Spain.

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. Instead, HOW is required for SXL to retain msl2 transcripts in the nucleus. Cooperation with SXL confers a sex-specific role to HOW. Our results uncover a novel function of SXL in nuclear mRNA retention and identify HOW as a mediator of this function.
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http://dx.doi.org/10.1101/gad.214999.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701196PMC
June 2013

Posttranscriptional control of X-chromosome dosage compensation.

Wiley Interdiscip Rev RNA 2011 Jul-Aug;2(4):534-45. Epub 2011 Feb 15.

Gene Regulation Programme, Centre for Genomic Regulation (CRG), UPF, Barcelona, Spain.

RNA regulation plays a major role in the generation of diversity at the molecular and cellular levels, and furnishes the cell with flexibility potential to adapt to changing environments. Often, the regulation by/of RNA dictates when, where, and how the information encoded in the nucleus is revealed. One example is the regulation of X-chromosome dosage compensation. In Drosophila, differences in X-linked gene dosage between males and females are compensated by the transcriptional upregulation of the single male X chromosome. Mechanisms of alternative splicing and translational control, among others, enforce dosage compensation in males while inhibiting this process in females. In this review, we discuss the posttranscriptional RNA regulatory mechanisms that ensure appropriate dosage compensation in Drosophila, drawing parallels with the mammalian system when appropriate.
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http://dx.doi.org/10.1002/wrna.75DOI Listing
March 2012

Identification of CUG-BP1/EDEN-BP target mRNAs in Xenopus tropicalis.

Nucleic Acids Res 2008 Apr 11;36(6):1861-70. Epub 2008 Feb 11.

CNRS, UMR 6061 Génétique et Développement, Université de Rennes 1, IFR 140 GFAS, 2 avenue du Pr Léon Bernard, CS 34317, 35043 Rennes Cedex and CNRS UMR 8080, Université Paris Sud, Orsay, France.

The early development of many animals relies on the posttranscriptional regulations of maternally stored mRNAs. In particular, the translation of maternal mRNAs is tightly controlled during oocyte maturation and early mitotic cycles in Xenopus. The Embryonic Deadenylation ElemeNt (EDEN) and its associated protein EDEN-BP are known to trigger deadenylation and translational silencing to several mRNAs bearing an EDEN. This Xenopus RNA-binding protein is an ortholog of the human protein CUG-BP1/CELF1. Five mRNAs, encoding cell cycle regulators and a protein involved in the notch pathway, have been identified as being deadenylated by EDEN/EDEN-BP. To identify new EDEN-BP targets, we immunoprecipitated EDEN-BP/mRNA complexes from Xenopus tropicalis egg extracts. We identified 153 mRNAs as new binding targets for EDEN-BP using microarrays. Sequence analyses of the 3' untranslated regions of the newly identified EDEN-BP targets reveal an enrichment in putative EDEN sequences. EDEN-BP binding to a subset of the targets was confirmed both in vitro and in vivo. Among the newly identified targets, Cdk1, a key player of oocyte maturation and cell cycle progression, is specifically targeted by its 3' UTR for an EDEN-BP-dependent deadenylation after fertilization.
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http://dx.doi.org/10.1093/nar/gkn031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330240PMC
April 2008

Identification of post-transcriptionally regulated Xenopus tropicalis maternal mRNAs by microarray.

Nucleic Acids Res 2006 7;34(3):986-95. Epub 2006 Feb 7.

CNRS UMR 6061, IFR 140, Functional Genetics, Agricultural and Health Sciences, Faculté de Médecine, Université de Rennes, 1 Rennes, France.

Cytoplasmic control of the adenylation state of mRNAs is a critical post-transcriptional process involved in the regulation of mRNAs stability and translational efficiency. The early development of Xenopus laevis has been a major model for the study of such regulations. We describe here a microarray analysis to identify mRNAs that are regulated by changes in their adenylation state during oogenesis and early development of the diploid frog Xenopus tropicalis. The microarray data were validated using qRT-PCR and direct analysis of the adenylation state of endogenous maternal mRNAs during the period studied. We identified more than 500 mRNAs regulated at the post-transcriptional level among the 3000 mRNAs potentially detected by the microarray. The mRNAs were classified into nine different adenylation behavior categories. The various adenylation profiles observed during oocyte maturation and early development and the analyses of 3'-untranslated region sequences suggest that previously uncharacterized sequence elements control the adenylation behavior of the newly identified mRNAs. These data should prove useful in identifying mRNAs with important functions during oocyte maturation and early development.
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http://dx.doi.org/10.1093/nar/gkj492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1361620PMC
February 2006

EDEN-BP-dependent post-transcriptional regulation of gene expression in Xenopus somitic segmentation.

Development 2004 Dec 17;131(24):6107-17. Epub 2004 Nov 17.

CNRS UMR 6061, IFR 97, Faculté de Médecine, Université Rennes 1, 2 avenue Léon Bernard, CS 34317, 35043 Rennes Cedex, France.

EDEN-BP is a Xenopus RNA-binding protein that triggers deadenylation [poly(A) tail shortening], and thereby translational repression and degradation, of a subset of maternal mRNAs soon after fertilization. We show here that this factor is expressed in the presomitic mesoderm of older embryos, the site where somitic segmentation takes place. Inhibiting EDEN-BP function using either antisense morpholino oligonucleotides or neutralizing antibodies leads to severe defects in somitic segmentation, but not myotomal differentiation. This is associated with defects in the expression of segmentation markers belonging to the Notch signalling pathway in the presomitic mesoderm. We show by a combination of approaches that the mRNA encoding XSu(H), a protein that plays a central role in Notch signalling, is regulated by the EDEN-BP pathway. Accordingly, XSu(H) is overexpressed in EDEN-BP knock-down embryos, and overexpressing XSu(H) causes segmentation defects. We finally give data indicating that, in addition to XSu(H), other segmentation RNAs are a target for EDEN-BP. These results show that EDEN-BP-dependent post-transcriptional regulation of gene expression is required for the process of somitic segmentation.
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http://dx.doi.org/10.1242/dev.01528DOI Listing
December 2004