The Biogenesis of Small Interfering RNA in Neurospora Crassa

RNA interference is a well-conserved gene silencing mechanism in eukaryotes. It regulates various biological processes including development, genome defense and heterochromatin formation. RNAi is initiated by the production of dsRNA, which is processed by Dicer to produce small interfering RNA (siRNA). In the filamentous fungus, Neurospora crassa, two types of siRNA have been characterized. One is involved in transgene-induced silencing, termed quelling; the other type is induced by DNA damage and functions to slow down protein translation after DNA damage. Both of these siRNAs originate from repetitive sequences in the Neurospora genome. We show that the components of the homologous recombination (HR) machinery are required to generate these types of small RNA specifically at repetitive regions. Furthermore, chromatin remodeling and DNA replication enzymes are required for efficient HR activity and small RNA production. Lastly, we show that the two small RNA pathways are mechanistically similar by demonstrating that quelling-induced siRNA can also be induced upon DNA damage. Our results suggest that the small RNA biogenesis machinery is recruited specifically to the repetitive loci after homologous recombination, which may result in the formation of aberrant DNA structures. dsRNA not only triggers the RNAi pathway, but also initiates a signaling cascade that results in activating the transcription of ~60 genes, including the RNAi components, in Neurospora. The function of the dsRNA activated genes suggests that RNAi is part of a broad ancient host defense response against viral and transposon infection. A genetic screen has been designed to identify the components involved in this dsRNA triggered transcriptional response; several mutants have been identified and characterized.