The Mechanism of Double-Stranded RNA Response in Neurospora

In eukaryotic cells, recognition of double-stranded RNA (dsRNA) by the enzyme Dicer initiates the RNA interference (RNAi) pathway, resulting in post-transcriptional gene silencing. Argonaute proteins play a critical role in this conserved pathway, which is present in protists, fungi, plants and animals. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that the production of dsRNA triggers the transcriptional induction of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for efficient gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 post-transcriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. A genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes (DRAGs) in Neurospora. Our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections. In order to understand the signaling mechanisms underlying this dsRNA response, we undertook a study of the dsRNA response elements (dsREs) in the promoter regions of qde-2 and other DRAGs. We demonstrate that different regions of the qde-2 promoter orchestrate early and late transcriptional induction in response to dsRNA. In the qde-2 promoter, a GC-rich element and downstream CAAT repeats were found to be important for the early response. In addition, the GC-rich dsRE was found in the promoters of other DRAGs, and was sufficient for dsRNA-induced transcriptional response. These results suggest that these DRAGs share the transcriptional induction pathway triggered by dsRNA. Finally, we demonstrate that QDE-2 contains an additional 10KDa N-terminal RGG domain, which is important for binding small interfering RNAs (siRNAs) and therefore required for its stability as well as efficient RNAi.