A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships(1,2). Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome(3), transcriptome(4) and proteome(5) data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes

Analyse fonctionnelle du promoteur de la cembranetriène- diol-synthase spécifique des trichomes de Nicotiana sylvestris

redaction en 2009Abstract In tobacco, secretory glandular trichomes (SGTs) are specifically producing volatiles compounds. This specificity is the consequence of the specific transcriptional expression of Terpenes synthases genes in SGTs. Promoters of those genes are of significant interest because they may be used in plant bioengineering to produce high value biomolecules (taxol, artemisinine) in SGTs. The promoter of a Cembrane-diol-sytnhase (P1.1NsTPSO2a), active in the SGTs heads, was isolated and transfected in transgenic N. sylvestris. Deletion analysis of P1.1NsTPSO2a shown 4c (100pb) and 400c (400pb) region required in trichome specificity. We next performed GUS fusion with the 4c region placed upstream of the minimum 35S promoter (-46) and demonstrate that 4c is sufficient to mimic the full-length promoter. Finally, we isolated a putative trichome transcription factor (NsZnf) that potentially binds the 400pb domain in yeast one-hybrid assay. NsZnf belongs to the GATA-ZincFinger III family. NsZnf possess a single zinc finger domain, a CCT domain and Tify domain. NsZnf nuclear localization was demonstrated by transient expression in N.benthamiana consistent with its role as transcription factor.Chez le tabac, les trichomes glandulaires sécréteurs (TGSs) sont des structures pluricellulaires où s'effectuent la synthèse et la sécrétion de nombreux composés volatiles. Cette production spécifique dans les TGSs est la conséquence de la spécificité de l'expression transcriptionnelle des gènes des Terpènes synthases. Les promoteurs de ces gènes sont intéressants parce qu'ils peuvent être utilisés en bioingénieurie pour produire des biomolécules à forte valeur ajoutées (taxol, l'artémisinine) dans TGSs. L'un d'eux, le promoteur de la Cembratriène-diol-synthase (P1.1NsTPSO2a), actif dans les cellules de têtes des TGSs, a été isolé et transfecté dans N.sylvestris. L'analyse par délétions de P1.1NsTPSO2a a montré que deux fragments, 4c (100pb) et 400c (360 pb) sont importants pour l'expression spécifique dans les têtes de trichomes. La région 4c a été placée en amont du promoteur 35S minimum (-46) et du gène rapporteur GUS. Nous avons démontré que 4c est nécessaire et suffisant pour une expression dans les têtes de TGSs. Le fragment 400c pourrait jouer un rôle dans la répression du P1.1NsTPSO2a dans les cellules épidermiques et dans certaines parties des racines. Enfin, nous avons isolé par crible simple-hybride dans la levure un facteur de transcription putatif du trichome (NsZnF) pouvant reconnaître le fragment 400c. NsZnf appartient à la famille des facteurs de transcription GATA-ZincFinger III. Cette protéine possède un doigt de zinc, un domaine CCT et un domaine TIFY. L'expression transitoire dans N.benthamiana montre une localisation nucléaire de NsZnf compatible avec son rôle de facteur de transcription

Analyse fonctionnelle du promoteur de la cembrane-triène-ol-synthase spécifique des trichomes de Nicotiana sylvestris

Chez le tabac, les tri chomes glandulaires sécréteurs (TGSs) sont des structures pluricellulaires où s'effectuent la synthèse et la sécrétion de nombreux composés volatiles. Cette production spécifique de composés volatiles dans les TGSs est la conséquence de la spécificité de l'expression transcriptionnelle des gènes des Terpènes synthases. Les promoteurs de ces gènes sont d'un grand intérêt parce qu'ils peuvent être utilisés en bioingénieurie pour produire des biomolécules à forte valeur ajoutées (taxol, l'artémisinine, sentalol) dans TGSs. Le promoteur de la Cembratriène diol-synthase (PIlNsTPS02a) a été isolé et transfecté dans N.sylvestris. Ce promoteur est très actif dans les cellules de têtes des TGSs. L'analyse par délétions du promoteur a montré que deux fragments, 4c (1 OOpb) et 400c (360 pb) sont importants pour l'expression spécifique dans les têtes de trichomes. La région de 4c a été placée en amont du promoteur 35S minimum (-46) et du gène rapporteur GUS. Nous avons ainsi démontré que cet élément est nécessaire et suffisant pour une expression dans les têtes de TGSs. Le fragment 400c pourrait jouer un rôle dans la répression du PIlNsTPS02a dans les cellules épidermiques et dans certaines parties des racines. Enfin, nous avons isolé par crible simple-hybride dans la levure un facteur de transcription putatif du trichome (NsZnF) qui pourrait reconnaître le fragment 400c. Cette protéine appartient à la famille des facteurs de transcription GAT A-ZincFinger. L'analyse bioinformatique a montré l'existence d'un doigt de zinc (domaine de fixation à l'ADN), d'un domaine CCT (localisation nucléaire) et un domaine TIF Y potentiellement impliqué dans l'homodimérisation. L'expression transitoire dans N.benthamiana montre une localisation nucléaire de NsZnf compatible avec son rôle de facteur de transcription. Ces travaux devraient permettre une meilleure compréhension des mécanismes de régulation transcriptionelle dans les TGSs et pourront servir au développement d'outils de production en bioingénieurie.ln Nicotiana.sylvestris, secretory glandular trichomes (SGTs) are tissues that specifically produce volatil compounds. This specificity is th consequence of the specific transcriptional expression of Terpene synthase genes in SGTs. Promoters of these genes are of significant interest becaus they may be used in plant bioengineering to produce high value biomolecules (taxol, artemisinine, sentalol) in SGTs. The promoter of the Cembram diol-synthase gene NsTPS02a was isolated and transfected in transgenic N.sylvestris. This PIlNsTPS02a promoter was strongly active in SGTs head cell: Using the GUS reporter gene, deletion analysis of the promoter in stable lines of Nicotiana showed that IWo regions, 4c (lOOpb) and 400c (360pb), a required for trichome-specific expression. When placed upstream of a minimum 35 pro.moter (-46) and the GUS gene, the 4c region provides trichome-specific expression profil. These experiments demonstrate that the 4c element is necessary and sufficient to mimic the full-length promote Based on deletion analysis, the 400c fragment could play a repressing role in the epidermal and root cells. Using yeast one-hybrid assay, we isolated putative trichome transcription factor, NsZnf that potentially binds the 400pb fragment. This protein belongs to the GAT A-ZincFinger famil)Computation analysis shows that the protein harbors a single zinc finger domain together with a CCT domain and a Tify domain often involved i homodimerization. Finally, we show by transient expression in N.benthamiana that NsZnf is localized in the nucleus consistently with its role a transcription factor. This work should help understanding transcription al regulation in SGTs and can help in designing tools for the production ( molecules in biotechnologyGRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Splicing Factor TRA2B Is Required for Neural Progenitor Survival

Alternative splicing of pre-mRNAs can rapidly regulate the expression of large groups of proteins. The RNA binding protein TRA2B (SFRS10) plays well-established roles in developmentally regulated alternative splicing during Drosophila sexual differentiation. TRA2B is also essential for mammalian embryogenesis and is implicated in numerous human diseases. Precise regulation of alternative splicing is critical to the development and function of the central nervous system; however, the requirements for specific splicing factors in neurogenesis are poorly understood. This study focuses on the role of TRA2B in mammalian brain development. We show that, during murine cortical neurogenesis, TRA2B is expressed in both neural progenitors and cortical projection neurons. Using cortex-specific Tra2b mutant mice, we show that TRA2B depletion results in apoptosis of the neural progenitor cells as well as disorganization of the cortical plate. Thus, TRA2B is essential for proper development of the cerebral cortex. (c) 2013 Wiley Periodicals, Inc

Modulation of nonsense mediated decay by rapamycin

Rapamycin is a naturally occurring macrolide whose target is at the core of nutrient and stress regulation in a wide range of species. Despite well-established roles as an inhibitor of cap-dependent mRNA translation, relatively little is known about its effects on other modes of RNA processing. Here, we characterize the landscape of rapamycin-induced post-transcriptional gene regulation. Transcriptome analysis of rapamycin-treated cells reveals genome-wide changes in alternative mRNA splicing and pronounced changes in NMD-sensitive isoforms. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD of certain transcripts. Rapamycin-treatment significantly reduces the levels of both endogenous and exogenous Premature Termination Codon (PTC)-containing mRNA isoforms and its effects are dose-, UPF1- and 4EBP-dependent. The PTC-containing SRSF6 transcript exhibits a shorter half-life upon rapamycin-treatment as compared to the non-PTC isoform. Rapamycin-treatment also causes depletion of PTC-containing mRNA isoforms from polyribosomes, underscoring the functional relationship between translation and NMD. Enhanced NMD activity also correlates with an enrichment of the nuclear Cap Binding Complex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin modulates global RNA homeostasis by NMD

IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) expression correlates with malignancy, but its role(s) in pathogenesis remains enigmatic. We interrogated the IGF2BP3-RNA interaction network in pancreatic ductal adenocarcinoma (PDAC) cells. Using a combination of genome-wide approaches, we have identified 164 direct mRNA targets of IGF2BP3. These transcripts encode proteins enriched for functions such as cell migration, proliferation, and adhesion. Loss of IGF2BP3 reduced PDAC cell invasiveness and remodeled focal adhesion junctions. Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) revealed significant overlap of IGF2BP3 and microRNA (miRNA) binding sites. IGF2BP3 promotes association of the RNA-induced silencing complex (RISC) with specific transcripts. Our results show that IGF2BP3 influences a malignancy-associated RNA regulon by modulating miRNA-mRNA interactions

A reference map of the human protein interactome

International audienceGlobal insights into cellular organization and function require comprehensive understanding of interactome networks. Similar to how a reference genome sequence revolutionized human genetics, a reference map of the human interactome network is critical to fully understand genotype-phenotype relationships. Here we present the first human “all-by-all” binary reference interactome map, or “HuRI”. With ~53,000 high-quality protein-protein interactions (PPIs), HuRI is approximately four times larger than the information curated from small-scale studies available in the literature. Integrating HuRI with genome, transcriptome and proteome data enables the study of cellular function within essentially any physiological or pathological cellular context. We demonstrate the use of HuRI in identifying specific subcellular roles of PPIs and protein function modulation via splicing during brain development. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian diseases. HuRI thus represents an unprecedented, systematic reference linking genomic variation to phenotypic outcomes