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International audience; ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

International audience; Hepatocellular carcinoma (HCC) is the major primary liver cancer. Glypican-3 (GPC3), one of the most abnormally expressed genes in HCC, participates in liver carcinogenesis. Based on data showing that GPC3 expression is posttranscriptionally altered in HCC cells compared to primary hepatocytes, we investigated the implication of microRNAs (miRNAs) in GPC3 overexpression and HCC. To identify GPC3-regulating miRNAs, we developed a dual-fluorescence FunREG (functional, integrated, and quantitative method to measure posttranscriptional regulations) system that allowed us to screen a library of 876 individual miRNAs. Expression of candidate miRNAs and that of GPC3 messenger RNA (mRNA) was measured in 21 nontumoral liver and 112 HCC samples. We then characterized the phenotypic consequences of modulating expression of one candidate miRNA in HuH7 cells and deciphered the molecular mechanism by which this miRNA controls the posttranscriptional regulation of GPC3. We identified five miRNAs targeting GPC3 3'-untranslated region (UTR) and regulating its expression about the 876 tested. Whereas miR-96 and its paralog miR-1271 repressed GPC3 expression, miR-129-1-3p, miR-1291, and miR-1303 had an inducible effect. We report that miR-1271 expression is down-regulated in HCC tumor samples and inversely correlates with GPC3 mRNA expression in a particular subgroup of HCC. We also report that miR-1271 inhibits the growth of HCC cells in a GPC3-dependent manner and induces cell death.CONCLUSION:Using a functional screen, we found that miR-96, miR-129-1-3p, miR-1271, miR-1291, and miR-1303 differentially control GPC3 expression in HCC cells. In a subgroup of HCC, the up-regulation of GPC3 was associated with a concomitant down-regulation of its repressor miR-1271. Therefore, we propose that GPC3 overexpression and its associated oncogenic effects are linked to the down-regulation of miR-1271 in HCC.

International audience; Besides the fact that miR-96 and miR-182 belong to the miR-182/183 cluster, their seed region (UUGGCA, nucleotides 2-7) is identical suggesting potential common properties in mRNA target recognition and cellular functions. Here, we used the mRNA encoding Glypican-3, a heparan-sulfate proteoglycan, as a model target as its short 3' untranslated region is predicted to contain one miR-96/182 site, and assessed whether it is post-transcriptionally regulated by these two microRNAs. We found that miR-96 downregulated GPC3 expression by targeting its mRNA 3'-untranslated region and interacting with the predicted site. This downregulatory effect was due to an increased mRNA degradation and depended on Argonaute-2. Despite its seed similarity with miR-96, miR-182 was unable to regulate GPC3. This differential regulation was confirmed on two other targets, FOXO1 and FN1. By site-directed mutagenesis, we demonstrated that the miRNA nucleotide 8, immediately downstream the UUGGCA seed, plays a critical role in target recognition by miR-96 and miR-182. Our data suggest that because of a base difference at miRNA position 8, these two microRNAs control a completely different set of genes and therefore are functionally independent.

An increasing number of arguments, including altered microRNA expression, support the idea that post-transcriptional deregulation participates in gene disturbances found in diseased tissues. To evaluate this hypothesis, we developed a method which facilitates post-transcriptional investigations in a wide range of human cells and experimental conditions. This method, called FunREG (functional, integrated and quantitative method to measure post-transcriptional regulation), connects lentiviral transduction with a fluorescent reporter system and quantitative PCR. Using FunREG, we efficiently measured post-transcriptional regulation mediated either by selected RNA sequences or regulatory factors (microRNAs), and then evaluated the contribution of mRNA decay and translation efficiency in the observed regulation. We demonstrated the existence of gene-specific post-transcriptional deregulation in liver tumour cells, and also reported a molecular link between a transcript variant abrogating HDAC6 (histone deacetylase 6) regulation by miR-433 and a rare familial genetic disease. Because FunREG is sensitive, quantitative and easy to use, many applications can be envisioned in fundamental and pathophysiological research.

International audience; The effects of Phytophthora cinnamomi root damage on water relations of chestnut (Castanea sativa) saplings were investigated. The relationship between root damage severity and impact on water relations was studied using a four compartment split-root system. Saplings were submitted to five inoculation levels and two watering conditions: normal (well-watered) or restricted watering. In well-watered saplings, stomatal conductance and whole plant transpiration rate were negatively correlated to the proportion of necrotic roots. Nevertheless, plant hydraulic conductance and leaf water potential were only affected above 90% necrotic roots. Interaction between root damage and water restriction was difficult to assess, since soil moisture decreased only slightly in infested compartments. However, saplings under restricted watering displayed lower stomatal conductance and transpiration values, regardless of root damage severity. Furthermore, the threshold of root damage leading to a decrease in leaf water potential was lower under restricted watering than under normal watering.; Effets de dégâts racinaires variables causés par Phytophthora cinnamomi sur le fonctionnement hydrique de jeunes châtaigniers. Les effets des dégâts racinaires causés par Phytophthora cinnamomi sur le fonctionnement hydrique de jeunes châtaigniers (Castanea sativa) ont été étudiés. Afin de relier les effets observés à la sévérité des dégâts racinaires, un système de "split-root " à quatre compartiments a été utilisé. Les arbres ont été soumis à cinq niveaux d'inoculation et à deux régimes hydriques (arrosage normal ou arrosage réduit). Chez les arbres normalement arrosés, une diminution linéaire de la conductance stomatique et de la transpiration avec la proportion de racines nécrosées a été observée. Cependant, la conductance hydraulique de l'arbre et le potentiel hydrique foliaire n'ont été affectés qu'à partir de 90 % de racines nécrosées. L'interaction entre les dégâts racinaires et la restriction en eau a été difficile à évaluer car l'humidité du sol a faiblement baissé dans les compartiments inoculés. Cependant, les arbres soumis a une restriction en eau avaient une conductance stomatique et une transpiration faibles quelle que soit la proportion de racines nécrosées. De plus, le seuil de dégâts racinaires entraînant une baisse de potentiel hydrique foliaire était plus faible chez les arbres soumis à une restriction en eau que chez les arbres normalement arrosés.