Jean-Christophe Rain

Functional and physical interaction between Bcl‐XL and a BH3‐like domain in Beclin‐1

The anti‐apoptotic proteins Bcl‐2 and Bcl‐XL 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‐XL is lost when the BH3 domain of Beclin‐1 or the BH3 receptor domain of Bcl‐XL is mutated. Mutation of the BH3 domain of Beclin‐1 or of the BH3 receptor domain of Bcl‐XL abolishes the Bcl‐XL‐mediated inhibition of autophagy triggered by Beclin‐1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin‐1 and Bcl‐2/Bcl‐XL, antagonizes autophagy inhibition by Bcl‐2/Bcl‐XL 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‐of‐function 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‐XL.

Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins

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 identified 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 significant 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 confirmed 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. Copyright

The Splicing ATPase Prp43p Is a Component of Multiple Preribosomal Particles

ABSTRACT 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.

Identification of the Helicobacter pylori anti‐σ28 factor

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 σ28 and σ54 factors of RNA polymerase respectively. The expression of flagellar genes is regulated, but no σ28‐specific effector was identified. It was also unclear whether H. pylori possessed a checkpoint for flagellar synthesis, and no gene encoding an anti‐σ28 factor, FlgM, could be identified by sequence similarity searches. To investigate the σ28‐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 σ28 factor and defined the C‐terminal part of HP1122 (residues 48–76) as the interaction domain. HP1122 interacts with region 4 of σ28 and prevents its association with the β‐region of H. pylori RNA polymerase. Thus, HP1122 presented the characteristics of an anti‐σ28 factor. This was confirmed in H. pylori by RNA dot‐blot hybridization and electron microscopy. The level of σ28‐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‐σ28 factor, FlgM, a major regulator of flagellum assembly. Potential anti‐σ28 factors were identified in Campylobacter jejuni, Pseudomonas aeruginosa and Thermotoga maritima by sequence homology with the C‐terminal region of HP1122.

A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation.

Inhibition of autophagy by TAB2 and TAB3

Autophagic responses are coupled to the activation of the inhibitor of NF‐κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ‐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.

In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein

Pre‐mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision. We previously developed a pre‐mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se. Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5′ splice site lead to pre‐mRNA export, whereas 3′ splice site mutations do not. A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre‐mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre‐mRNA. In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially. Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.

Functional Conservation of the Transportin Nuclear Import Pathway in Divergent Organisms

ABSTRACT 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 signal in mammalian cells. Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro. We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs. Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution.

Synthesis and Biological Evaluation of 9-Oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile Analogues as Potential Inhibitors of Deubiquitinating Enzymes

High‐throughput screening highlighted 9‐oxo‐9H‐indeno[1,2‐b]pyrazine‐2,3‐dicarbonitrile (1) as an active inhibitor of ubiquitin‐specific proteases (USPs), a family of hydrolytic enzymes involved in the removal of ubiquitin from protein substrates. The chemical behavior of compound 1 was examined. Moreover, the synthesis and in vitro evaluation of new compounds, analogues of 1, led to the identification of potent and selective inhibitors of the deubiquitinating enzyme USP8.

Building protein-protein networks by two-hybrid mating strategy.

Publisher Summary The basic concept of the yeast two-hybrid system is to detect the interaction between two proteins via transcriptional activation of a reporter gene. A classical eukaryotic transcription activator contains a domain that specifically binds to DNA sequences and a domain that recruits the transcription machinery. The yeast two-hybrid system can detect interactions between two known proteins or polypeptides and can also search for unknown partners of a given protein. The yeast two-hybrid system is an indirect genetic assay. Intrinsic limitations of the yeast two-hybrid system include reliance on complex transcriptional activation of reporter genes. Incorrect folding, inappropriate subcellular localization (bait and prey proteins must interact in a nuclear environment), or degradation of chimeric proteins and absence of certain types of posttranslational modifications in yeast could lead to false negatives. Other properties of the assay may lead to the selection of false positives. Confirmation of interactions should be obtained via various independent genetic, biochemical or functional assays, such as copurification in complexes, colocalization, or demonstration of a functional association.