The DNA/RNA-dependent RNA polymerase QDE-1 generates aberrant RNA and dsRNA for RNAi in a process requiring replication protein A and a DNA helicase

• Published in: PLoS biology Vol. 8; no. 10; p. e1000496
• Main Authors: Lee, Heng-Chi, Aalto, Antti P, Yang, Qiuying, Chang, Shwu-Shin, Huang, Guocun, Fisher, Daniel, Cha, Joonseok, Poranen, Minna M, Bamford, Dennis H, Liu, Yi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01-10-2010; Public Library of Science (PLoS)
• Subjects: Animals; Biochemistry; Biomedical research; Deoxyribonucleic acid; DNA; DNA Helicases - genetics; DNA Helicases - metabolism; DNA replication; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fungi; Gene expression; Gene Expression Regulation, Fungal; Humans; Molecular Biology/mRNA Stability; Neurospora; Neurospora crassa - genetics; Neurospora crassa - metabolism; Physiological aspects; Plant Biology; Proteins; Replication Protein A - genetics; Replication Protein A - metabolism; Ribonucleases - genetics; Ribonucleases - metabolism; Ribonucleic acid; RNA; RNA - genetics; RNA - metabolism; RNA Interference; RNA polymerase; RNA polymerases; RNA Replicase - genetics; RNA Replicase - metabolism; RNA, Double-Stranded - genetics; RNA, Double-Stranded - metabolism; United States; Gene Expression Regulation, Fungal; RNA; Humans; Fungal Proteins; Ribonucleases; DNA Helicases; Animals; RNA Interference; Neurospora crassa; RNA Replicase; RNA, Double-Stranded; DNA, Single-Stranded; Replication Protein A
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.1000496

The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.