The Mechanism of RNA Interference in Neurospora

In the canonical RNA interference (RNAi) pathway, small-interfering RNA (siRNA) duplexes generated by Dicer are incorporated into the RNA-induced-silencing complex (RISC), and subsequently converted to single-stranded siRNA. Generation of single stranded siRNA is a pre-requisite for recognition and cleavage of the target mRNA by Argonaute. In biochemical experiments, Argonaute generates single-stranded siRNA by cleaving the passenger strand of the siRNA duplex. Mutational analysis of Neurospora homologue of Argonaute-2, known as Quelling Deficient -2 (QDE-2), revealed that the endonuclease activity of QDE-2 is required for the generation of singlestranded siRNA in vivo. Further biochemical studies to understand the mechanism for removal of the nicked passenger strand from siRNA duplex, led to the identification of a novel QDE-2 interacting protein (QIP) with a putative exonuclease domain. Disruption of qip led to the impairment of RNAi and most of the siRNAs were accumulated in nickedduplex form. Furthermore, QIP functions as an exonuclease to remove the cleaved passenger strand in a QDE-2 dependent manner. Thus, the cleavage of the passenger strand by QDE2 and its subsequent removal by QIP are critical biochemical steps in Neurospora RNAi pathway. Quelling, an RNAi related phenomenon in Neurospora, is induced by multiple copies of transgene. It was proposed that QDE-1 (a RNA dependent RNA polymerase, RdRp) and QDE-3 (a RecQ helicase) functions in quelling pathway by generating double-stranded RNA (dsRNA) from transgenes. To further understand the importance of QDE-1 and QDE-3, quelling assays were performed in the qde-1ko and qde-3ko strains. In contrast to previous results, the requirement of QDE-1 and QDE-3 was bypassed when the transgene copy number was high. Moreover, gene silencing analyses using strains lacking all potential RdRps suggested that unlike in C.elegans and Arabidopsis, the amplification of secondary dsRNA or siRNA is largely absent in Neurospora. The search for potential regulatory mechanisms of RNAi components in Neurospora led to the identification of a dsRNA response pathway. Two key components of the Neurospora RNAi pathway, qde2 and dicer like protein-2 (dcl-2), are induced by dsRNA at transcriptional and posttranscriptional level. The induction of QDE-2 is required for efficient gene silencing, indicating the importance of this regulatory mechanism in RNAi pathway.