Kinetic analysis of RNA interference in nuclear and cytoplasmic compartments in mammalian cells
RNA interference (RNAi) is a mechanism by which double stranded RNA trigger the sequence specific degradation of RNA. In mammalian cells, RNAi can be induced by short interfering RNA (siRNA). RNAi is carried out by the RISC complex (RNA induced silencing complex) that contains at least the RNA guide sequence and the Ago2 nuclease, enabling target identification and cleavage, respectively. In this thesis we focused on the kinetic analysis of the mRNA degradation induced by siRNA in mammalian cells. We measured the half-lives of the β-globin and lymphotoxin-α (LTα) silenced mRNA and showed that these kinetics reflected in part the efficiency of silencing of the corresponding siRNA. However, we also observed for two efficient siRNA, that the degradation kinetics accelerate between 16 h and 24 h after transfection. In addition, the observation that the same siRNA induce different inhibitions for the two LTα messengers has led us to propose an interaction between translation and RNA interference. We have investigated the impact of RNA interference on the nuclear mRNA metabolism. We thus demonstrated that siRNA induce the degradation of nuclear RNA with an efficacy that depends on a kinetic competition between interference and processing reactions involving the targeted transcript (splicing, transport). Moreover, we observed that the degradation of a messenger RNA could be accompanied by the accumulation of its precursors, probably due to a higher RNA synthesis. Thus, interference can shed light on new regulations of the nuclear mRNA metabolism.