Characterization of Systemic RNAi Mutants in Caenorhabditis Elegans

Abstract

RNA interference (RNAi) is sequence-specific gene silencing triggered by double-stranded (ds)RNA. When dsRNA is expressed or introduced into one cell and is transported to and initiates RNAi in other cells, it is called systemic RNAi. Systemic RNAi enables easy and efficient whole-animal gene silencing in C. elegans. While the specificity and potency of RNAi offers tremendous therapeutic potential to treat genetic and infectious disease, effective delivery to human cells remains a major challenge. Understanding systemic RNAi transport pathways in C. elegans may help identify strategies for effective delivery in mammals. The Hunter lab conducted a genetic screen in C. elegans that identified five genes, sid-1 through -5, that are specifically required for systemic RNAi. Using whole genome sequencing, I identified sid-4 as nck-1, a non-receptor tyrosine kinase adaptor protein. Interestingly, sid-3 is homologous to ACK tyrosine kinases that physically interact with NCK homologs. SID-3 and SID-4 are both broadly expressed and show similar subcellular localization patterns. My analysis showed that sid-4, like sid-3, is a dose-dependent import mutant. Although sid-3 and sid-4 mutants showed similar patterns of RNAi resistance, the sid-3; sid-4 double mutant was significantly more RNAi defective than either single mutant, indicating that these proteins do not function only in a simple linear pathway. Consistent with their identity as conserved signaling proteins, their activity is required to mediate temperature-dependent systemic RNAi efficiency effects. Interestingly, both mutants form dauers at elevated temperatures (27°C), suggesting that RNA mobility may regulate growth and development. A likely model is that SID-3/SID-4 act to modulate the activity of the conserved dsRNA conducting channel, SID-1.