Investigating the Role of the IP₃ Signalling Pathway in RNA Interference in C. elegans
Both inositol 1,4,5-trisphosphate (IP₃) mediated signalling and RNA interference are widespread processes with fundamental roles in animal cell function. In the nematode C. elegans these two pathways have been shown to intersect such that IP₃ signalling mutants display an altered exogenous RNAi response. IP₃ is a key second messenger in the transduction of intracellular signals. Produced by the hydrolysis of membrane phospholipid PIP₂ by phospholipase C (PLC), IP₃ triggers Ca²⁺ release from internal stores by binding to the IP₃ receptor, a ligand gated calcium channel in the ER, thus triggering downstream calcium signalling pathways. It has been previously shown that loss of function in the IP₃ receptor (ITR-1) or in PLCβ (EGL-8) results in an enhanced response to exogenous dsRNA, with a more robust silencing response than that seen in WT worms. Conversely the potentiation of IP₃ signalling by the loss of function IPP-5, a phosphatase which catalyses the hydrolysis of IP₃, results in resistance to RNAi. In order to better understand the context of the IP₃ signalling events which are regulating the RNAi response I sought to identify the upstream activator of EGL-8 in this mechanism. EGL-8 is canonically activated by Gαq homologue ELG-30. However, by screening the RNAi responses of Gα signalling mutants using a range of assays, EGL-30 was ruled out as an activator of EGL-8 in the pathway regulating RNAi, since EGL-30 loss of function does not result in an enhanced RNAi response. The Gαo/i homologue, GOA-1, was identified as the most likely activator of EGL-8 due to the strongly enhanced RNAi response in goa-1 loss of function mutants in a number of assays. Other Gα subunits and known regulators of GOA-1 and EGL-30 signalling were also analysed. The RNAi response is a multistep process in which the exogenous RNAi pathway must compete with the closely related and interlinked endogenous small RNA pathways for resources. With the aim of better understanding how changes in IP₃ signalling might be influencing the RNAi response, small RNA sequencing was performed to look for evidence of change in the endogenous small RNA pathways of itr-1 mutants, and followed up with qPCR. However, no substantial changes to the endogenous small RNA pathways were found. Downstream of the production of primary siRNAs from exogenous dsRNA, separate argonautes mediate cytoplasmic and nuclear RNAi responses. I utilised an established assay to test for effects of IP₃ signalling on nuclear RNAi. The results suggest no specific alterations to the nuclear RNAi pathway in these mutants.