GSE129063
Recognition and processing of long double-stranded RNA in mammalian cells
In A. thaliana, C. elegans and Drosophila, long dsRNA accumulation during virus infection, transposon activation or transgene expression, elicits a potent RNAi response: long dsRNA processing into siRNAs by Dicer, loading into Argonaute and assembly of the RNA-induced silencing complex to slice complementary RNA. In mammals, recognition of long dsRNA by type I interferon (IFN) factors, results in a mitigated RNAi response in some cell types. Here we uncover key mechanisms associated with recognition and processing of long dsRNA by both pathways. First, we demonstrate that exogenous long dsRNA rapidly congregates into granules recruiting key RNA silencing and type I IFN factors, suggesting an interaction between these pathways. Second, we confirm that inactivation of PKR, unleashes a potent RNAi response on transgene and endogenous mRNA targets in HEK293T cells. Finally, we observe different Dicer processing modes and uncovered a role for TRBP as regulator of RNAi in a template-dependent manner. Overall design: Total and Ago-pulldown sRNA sequencing of different HEK293T knock-out backgrounds transfected with various long dsRNA templates to elucidate the mechanism of mammalian RNAi and genetically dissect which proteins are involved