Interférence par ARN | EPFL Graph Search

Un ARN interférent est un acide ribonucléique (ARN) simple ou double brin dont l'interférence avec un ARN messager spécifique conduit à sa dégradation et à la diminution de sa traduction en protéine. Dans la mesure où l'ARN joue un rôle crucial dans l'expression des gènes, l'ARN interférent permet de bloquer celle-ci en rendant « silencieux » tel ou tel gène. Ce phénomène a été découvert dans les années 1990, valant à Andrew Z. Fire et Craig C. Mello le prix Nobel de physiologie et de médecine en 2006. Il serait vraisemblablement un produit de l'évolution permettant aux organismes de se défendre contre l'introduction de génomes étrangers, notamment viraux, ou encore permettant de moduler l'expression des gènes. Chez les mammifères, l'ARN interférent doit mesurer environ 22 nucléotides pour être spécifique, autrement la formation d'un ARN double brin active la réponse immunitaire antivirale interféron, causant la dégradation non spécifique de tous les ARN. Historique L'i

Characterization of RGS-7 as a regulator of forces involved in C. elegans early cell divisions

Simon Germann

Proper regulation of cell division is crucial to most organisms. Asymmetric division and unequal cleavage are one of the many mechanisms by which spatial regulation of cell division might be achieved. Indeed, by varying its polarity cues or by modulating the size of its daughters, a cell can potentially influence its cellular neighborhood and possibly act upon it. In C. elegans, asymmetric cell division occurs at early stages of development, and is induced by an asymmetric positioning of the spindle during mitosis. RGS-7, a member of the RGS protein family, is a potential candidate as a regulator of this process, and was shown to affect the balance of pulling forces responsible for the asymmetric position of the spindle, possibly acting through Gα and/or Gβ, which are heterotrimeric G-protein complex subunits involved in force generation in C. elegans early cell divisions. However, many aspects of the role this protein may have in this process remain to be elucidated. Using available rgs-7 mutants, RNA interference, spindle severing experiments and immunofluorescence assays, this study highlights the potential functions RGS-7 may provide to a dividing C. elegans embryo

2008

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

The Notch signaling pathway is a key regulator of cell fate decisions in embryonic development and in adult tissue homeostasis. Mounting evidence suggests that Notch signaling is frequently deregulated in human neoplasms, where depending upon the cellular context it can function both as an oncogene or a tumor suppressor. A plethora of studies shed light on how Notch crosstalks with signaling pathways downstream to manifest either its oncogenic or tumor suppressive activity. However, regulatory networks upstream of the Notch signaling pathway are still poorly understood. Since protein kinases are well-known regulators of a majority of cell signaling pathways, the aim of the current study was to identify novel positive and negative regulatory kinases that modulate Notch signaling. Using a HeLa cell based co-culture assay to read Notch-dependent luciferase activity, a small interference RNA (siRNA) library against the human kinase genome was previously tested in a high-throughput manner. Validation of top candidate kinases from the screening using both siRNA as well as pharmacological inhibitors led to further elimination of false positives and identification of Protein Kinase B (AKT2) and Calmodulin Kinase II (CaMKII) as key positive while Janus kinases (JAKs) as key negative regulators of the Notch signaling pathway. In the first part of the study, we analyzed the positive regulation of AKT2 and CaMKII kinases on Notch signaling in the T-cell acute lymphoblastic leukemia (T-ALL) cells where Notch is oncogenic. It was shown that pharmacological inhibition of either AKT2 or CaMKII kinase in T-ALL cell lines in vitro abrogated the expression of active Notch1 intracellular domain (N1-ICD) as well as of Notch target genes. Moreover, inhibition of these kinases blocked proliferation of T-ALL cells. In the second part of the study, we analyzed the negative regulation of JAK kinases on Notch signaling using a distinct physiological context where Notch is tumor suppressive. It was shown that pharmacological inhibition of JAK kinases led to an induction of Notch receptor transcription and of Notch target genes in the human primary epidermal keratinocytes (HPEK) as well as in the skin squamous cell carcinoma (SCC) cell lines. In addition, the pharmacological inhibition of JAK kinases induced a block in proliferation, cell cycle arrest and differentiation in HPEKs and skin SCCs by increased expression of early differentiation markers. Suppression of Notch signaling in primary keratinocytes counteracted the differentiation-inducing effect of JAK inhibition. Since JAK inhibition affected Notch transcription, global gene expression profiling using RNA sequencing was done to identify transcription factors involved in an indirect regulation of JAK kinases on the Notch cascade. EGR1 and EGR2 belonging to the early growth response family of transcription factors were significantly modulated downstream of JAK inhibition. In vivo, topical inhibition of JAK kinases provided marked resistance against chemically induced cutaneous carcinogenesis.Taken together, our data reveals a key role of AKT2 and CaMKII kinases in positive regulation while of JAK kinases in the negative regulation of Notch signaling, of potential relevance for combinatory approaches for cancer therapy. Pharmacological inhibitors against these kinases could be tested for their ability to modulate Notch signaling and may prove highly beneficial in the therapy of Notch-driven cancers.

EPFL2016

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

EPFL2016