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

Yi Liu, Heng Chi Lee, Antti P. Aalto, Qiuying Yang, Shwu Shin Chang, Guocun Huang, Daniel Fisher, Joonseok Cha, Minna M. Poranen, Dennis H. Bamford

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

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.© 2010 Lee et al.

Original languageEnglish (US)
Article numbere1000496
JournalPLoS biology
Volume8
Issue number10
DOIs
StatePublished - Oct 2010

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Agricultural and Biological Sciences(all)

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    Liu, Y., Lee, H. C., Aalto, A. P., Yang, Q., Chang, S. S., Huang, G., Fisher, D., Cha, J., Poranen, M. M., & Bamford, D. H. (2010). 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. PLoS biology, 8(10), [e1000496]. https://doi.org/10.1371/journal.pbio.1000496