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.
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.
Lee
Heng-Chi
HC
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
Aalto
Antti P
AP
Yang
Qiuying
Q
Chang
Shwu-Shin
SS
Huang
Guocun
G
Fisher
Daniel
D
Cha
Joonseok
J
Poranen
Minna M
MM
Bamford
Dennis H
DH
Liu
Yi
Y
D000818:Animals; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D005656:Fungal Proteins; C406184:QDE-2 protein, Neurospora crassa; D012330:RNA, Double-Stranded; D051716:Replication Protein A; D012313:RNA; C119303:QDE1 protein, Neurospora crassa; D012324:RNA-Dependent RNA Polymerase; D012260:Ribonucleases; D004265:DNA Helicases; C402647:QDE-3 protein, Neurospora crassa