Double knockdown of the Rheb gene in mammalian cells using RNA interference

Abstract

The insulin/mTOR pathway has been implicated in a variety of cancers and activation of mTOR (mammalian Target of Rapamycin) is regulated by an upstream G‐protein, Rheb (Ras Homolog Enriched in Brain). In mammalian systems two Rheb genes exist – Rheb1 and Rheb2 (RhebL1). Although significant sequence homology is shared, we hypothesize that Rheb2 may have distinct functions of its own. To elucidate the function of Rheb2, RNA interference was utilized to silence the expression of the Rheb genes in vitro. Rheb1 and Rheb2 were expressed individually as GFP‐fused proteins in mammalian cell lines and co‐transfected with Rheb specific siRNAs. Endogenous Rheb1/2 was silenced either individually or in a dual manner. Effect of silencing on growth profiles of cells was analyzed via MTT cell viability assays and the mTOR activation status was studied using immunoblotting and probing for phophorylation status of downstream effectors. This study shows for the first time that silencing Rheb2 impacts the mTOR pathway in a different way than Rheb1 and reinforces our hypothesis that Rheb2 may have specific roles not attributed to Rheb1. This is also the first report to simultaneously study the endogenous versions of Rheb1 and Rheb2 rather than over‐expressed forms of the same and provides a true profile of natural signaling conditions within a cell. This work was supported by a CSUPERB‐Doris A. Howell Research Award to AB.