Papers by Jean-christophe Rain
Annales de l'Institut Pasteur / Actualités, 2002
HA-tagged full-length TAB2 or TAB3. The indicated constructs were transfected into HeLa cells. Af... more HA-tagged full-length TAB2 or TAB3. The indicated constructs were transfected into HeLa cells. After 24 hours, cells were cultured for 2 hours in Earle's balanced salt solution (starvation) or in complete medium supplemented with 1 μM rapamycin or 30 μM pifithrin α (PFTα), followed by cell lysis, immunoprecipitation of His-BCN1, SDS-PAGE and immunodetection of HA-tagged proteins. GAPDH levels were monitored to ensure equal loading. Moreover, the input was analyzed to ensure the equal expression of His-BCN1, HA-TAB2 and HA-TAB3.
Molecular and cellular biology, 1998
Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding pr... more Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins [hnRNP]) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm. hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro. Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned. To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif. NAB35 also interacts with hTRN1 and functions as a nuclear localization ...
Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human im... more Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3′ processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.
Methods in Enzymology, 2002
... Gene 242, 369 (2000). 12 M. Fromont-Racine, AE Mayes, A. Brunet-Simon, J.-C. Rain, A. Colley,... more ... Gene 242, 369 (2000). 12 M. Fromont-Racine, AE Mayes, A. Brunet-Simon, J.-C. Rain, A. Colley, I. Dix, L. Decourty, N. Joly, E Ricard, JD Beggs, and P. Legrain, Yeast Comparative Functional Genomics 17, 95 (2000). 13 j. C ...
Yeast, 2000
A set of seven structurally related Sm proteins forms the core of the snRNP particles containing ... more A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identi®ed a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally signi®cant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are con®rmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein±protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.
The EMBO Journal, 2007
The anti-apoptotic proteins Bcl-2 and Bcl-X L bind and inhibit Beclin-1, an essential mediator of... more The anti-apoptotic proteins Bcl-2 and Bcl-X L bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114-123). The physical interaction between Beclin-1 and Bcl-X L is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-X L is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-X L abolishes the Bcl-X L -mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-X L , antagonizes autophagy inhibition by Bcl-2/Bcl-X L and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-offunction mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-X L .
The EMBO Journal, 2009
The nuclear factor Acinus has been suggested to mediate apoptotic chromatin condensation after ca... more The nuclear factor Acinus has been suggested to mediate apoptotic chromatin condensation after caspase cleavage. However, this role has been challenged by recent observations suggesting a contribution of Acinus in apoptotic internucleosomal DNA cleavage. We report here that AAC-11, a survival protein whose expression prevents apoptosis that occurs on deprivation of growth factors, physiologically binds to Acinus and prevents Acinusmediated DNA fragmentation. AAC-11 was able to protect Acinus from caspase-3 cleavage in vivo and in vitro, thus interfering with its biological function. Interestingly, AAC-11 depletion markedly increased cellular sensitivity to anticancer drugs, whereas its expression interfered with drug-induced cell death. AAC-11 possesses a leucine-zipper domain that dictates, upon oligomerization, its interaction with Acinus as well as the antiapoptotic effect of AAC-11 on drug-induced cell death. A cell permeable peptide that mimics the leucine-zipper subdomain of AAC-11, thus preventing its oligomerization, inhibited the AAC-11-Acinus complex formation and potentiated drug-mediated apoptosis in cancer cells. Our results, therefore, show that targeting AAC-11 might be a potent strategy for cancer treatment by sensitization of tumour cells to chemotherapeutic drugs.
The EMBO Journal, 2011
Autophagic responses are coupled to the activation of the inhibitor of NF-jB kinase (IKK). Here, ... more Autophagic responses are coupled to the activation of the inhibitor of NF-jB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFb-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.
Retrovirology, 2008
Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a... more Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex -the reverse transcription complex (RTC) -consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein -the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the Cterminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.
Molecular Microbiology, 2001
Flagellar motility is essential for colonization of the human gastric mucosa by Helicobacter pylo... more Flagellar motility is essential for colonization of the human gastric mucosa by Helicobacter pylori. The flagellar filament is composed of two subunits, FlaA and FlaB. Transcription of the genes encoding these proteins is controlled by the s 28 and s 54 factors of RNA polymerase respectively. The expression of flagellar genes is regulated, but no s 28 -specific effector was identified. It was also unclear whether H. pylori possessed a checkpoint for flagellar synthesis, and no gene encoding an anti-s 28 factor, FlgM, could be identified by sequence similarity searches. To investigate the s 28 -dependent regulation, a new approach based on genomic data was used. Two-hybrid screening with the H. pylori proteins identified a protein of unknown function (HP1122) interacting with the s 28 factor and defined the C-terminal part of HP1122 (residues 48-76) as the interaction domain. HP1122 interacts with region 4 of s 28 and prevents its association with the b-region of H. pylori RNA polymerase. Thus, HP1122 presented the characteristics of an anti-s 28 factor. This was confirmed in H. pylori by RNA dot-blot hybridization and electron microscopy. The level of s 28 -dependent flaA transcription was higher in a HP1122-deficient strain and was decreased by the overproduction of HP1122. The overproduction of HP1122 also resulted in H. pylori cells with highly truncated flagella. These results demonstrate that HP1122 is the H. pylori anti-s 28 factor, FlgM, a major regulator of flagellum assembly. Potential anti-s 28 factors were identified in Campylobacter jejuni, Pseudomonas aeruginosa and Thermotoga maritima by sequence homology with the C-terminal region of HP1122.
Molecular and Cellular Biology, 2005
Prp43p is a putative helicase of the DEAH family which is required for the release of the lariat ... more Prp43p is a putative helicase of the DEAH family which is required for the release of the lariat intron from the spliceosome. Prp43p could also play a role in ribosome synthesis, since it accumulates in the nucleolus. Consistent with this hypothesis, we find that depletion of Prp43p leads to accumulation of 35S pre-rRNA and strongly reduces levels of all downstream pre-rRNA processing intermediates. As a result, the steady-state levels of mature rRNAs are greatly diminished following Prp43p depletion. We present data arguing that such effects are unlikely to be solely due to splicing defects. Moreover, we demonstrate by a combination of a comprehensive two-hybrid screen, tandem-affinity purification followed by mass spectrometry, and Northern analyses that Prp43p is associated with 90S, pre-60S, and pre-40S ribosomal particles. Prp43p seems preferentially associated with Pfa1p, a novel specific component of pre-40S ribosomal particles. In addition, Prp43p interacts with components of the RNA polymerase I (Pol I) transcription machinery and with mature 18S and 25S rRNAs. Hence, Prp43p might be delivered to nascent 90S ribosomal particles during pre-rRNA transcription and remain associated with preribosomal particles until their final maturation steps in the cytoplasm. Our data also suggest that the ATPase activity of Prp43p is required for early steps of pre-rRNA processing and normal accumulation of mature rRNAs.
Methods, 2009
Integrase LEDGF/p75 Transportin-SR2 VBP1 Yeast two-hybrid Virus-host interactions SNF5 Chromatin ... more Integrase LEDGF/p75 Transportin-SR2 VBP1 Yeast two-hybrid Virus-host interactions SNF5 Chromatin tethering Retrovirus integration
Mechanisms of Development, 2009
The POU-V transcription factor Oct4 is a master regulator of self-renewal and pluripotency in emb... more The POU-V transcription factor Oct4 is a master regulator of self-renewal and pluripotency in embryonic stem (ES) cells as well
Journal of Virology, 2004
We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies vi... more We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcriptionreplication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's,
Journal of Proteomics, 2014
Intensive methodological developments and technology innovation have been devoted to protein-prot... more Intensive methodological developments and technology innovation have been devoted to protein-protein interaction studies over 20years. Genetic indirect assays and sophisticated large scale biochemical analyses have jointly contributed to the elucidation of protein-protein interactions, still with a lot of drawbacks despite heavy investment in human resources and technologies. With the most recent developments in mass spectrometry and computational tools for studying protein content of complex samples, the initial goal of deciphering molecular bases of biological functions is now within reach. Here, we described the various steps of this process and gave examples of key milestones in this scientific story line. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.
Journal of Molecular Biology, 2007
Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human im... more Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3′ processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.
Journal of Molecular Biology, 2008
Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a... more Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex -the reverse transcription complex (RTC) -consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein -the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the Cterminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.
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Papers by Jean-christophe Rain