The Function and Mechanism of RNA Interference in Neurospora

RNA interference (RNAi) is a conserved gene silencing mechanism important for various biological processes, including developmental timing, genome defense, and heterochromatin formation. RNAi is triggered by double stranded RNA (dsRNA), which is processed by Dicer to siRNA. siRNA is loaded onto RNA-induced silencing complex (RISC), in which an Argonaute family protein, guided by a siRNA, mediates the cleavage of homologous RNAs. In the filamentous fungus Neurospora, we show that dsRNA not only trigger RNAi, it also transcriptionally activates several key components of RNAi pathway, including qde-2 (an Argonaute) and dcl-2 (a Dicer). A genome wide identification of dsRNA activated genes suggests that RNAi is part of a broad ancient host-defense response against viral and transposon infections. Our research on qde-2 regulation also suggests a role of RNAi during DNA damage. We show that DNA damage induces qde-2 expression, and the purification of QDE-2 bound RNAs identifies a novel class of small RNAs named qiRNAs. qiRNAs are averaged 21 nt in length and are mostly derived from ribosomal DNA (rDNA) locus. Importantly, qiRNA biogenesis requires RNAi components and RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs during DNA repair. Further analysis suggests that the qiRNA contributes to the DNA damage checkpoints by inhibiting protein translation after DNA damage. To trigger RNAi against transgenes, it has been proposed that transgene- specific aberrant RNA (aRNA) is made and converted into dsRNA by RNA dependent RNA polymerase (RdRP). How aRNA is produced and specifically recognized by RdRP is not known. We show that QDE-1, a RdRP is also the DNA-dependent RNA polymerase (DdRP) that produces aRNA from ssDNA. QDE-1 is recruited to ssDNA by Replication Protein A (RPA) and QDE-3 (an RecQ helicase), both of them are also essential for aRNA production. Moreover, QDE-1 can produce dsRNA from ssDNA, a process facilitated by RPA. Our results provide a molecular mechanism of aRNA production in RNAi pathway.