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

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.

Figure 1. Recombinant QDE-1 exhibits both RdRP and DdRP activities.

(A) In vitro RNA polymerase assay using the 176 nt ssDNA or ssRNA templates and recombinant QDE-1ΔN, catalytically inactive QDE-1ΔN^DA or bacteriophage φ6 RdRP. ³²P-labeled reaction products were resolved on a 3% TBE agarose gel. The template lanes contain the corresponding 5′-terminally ³²P-labeled ssDNA or ssRNA templates. In the other lanes, the templates were not labeled. (B) Characterization of the QDE-1ΔN products from DdRP and RdRP assays by various nuclease treatments. The upper panels show images of ethidium bromide (EtBr) stained gels, and the lower panels are autoradiographs of the same gels. CTL, control. (C and D) In vitro polymerase assays using QDE-1ΔN and a 3 kb ssRNA or ssDNA template. In (C), both ethidium bromide (EtBr) stained images and autoradiographs of the gels are shown. The reactions were initiated by the addition of QDE-1ΔN and samples were collected at the indicated time points. In (D), the values in arbitrary units of incorporated radioactivity were quantified for each product from three independent experiments. The error bars indicate standard errors of the mean. Note the different activity scales used for the ssDNA- or ssRNA-templated assays.

Figure 2. RPA is required for aRNA and qiRNA production and for quelling.

(A) Western blot analyses of QDE-2 induction by histidine in the wild type (WT), rpa-3 knock-out (rpa-3^ko), rpa-1 knock-down (dsrpa-1), and rpa-2 knock-down (dsrpa-2) strains. Two independent rpa-3^ko strains were used. (B) Northern blot analysis of qiRNA production in the wild type (WT) and rpa-3 ^ko strains after histidine treatment. Histidine induces DNA damage in Neurospora. (C) qRT-PCR showing the production of DNA damage-induced aRNA from the rDNA locus after histidine treatment in wild type and rpa-3^ko strains. (D) The results of the quelling assay showing the silencing efficiency of albino-1 (al-1) gene in the wild-type, qde-3^ko, and rpa-3^ko strains. Percentage and actual numbers (shown in parenthesis) of al-1 silenced transformants are shown.

Figure 3. RPA promotes ssDNA-directed synthesis of dsRNA by QDE-1.

(A) In vitro DdRP assay using circular ssDNA from bacteriophages M13 or φX174 as templates. The nature of the products was characterized by various nuclease treatments and native agarose gel (0.6%) electrophoresis. (B) In vitro DdRP assay using full-length recombinant QDE-1 with various concentrations of the RPA complex. The 175 nt ssDNA template was pre-incubated with RPA before adding QDE-1, and the products were resolved by 6% urea containing polyacrylamide gel. The DNA/RNA hybrid refers to the single-stranded, labeled RNA products of the denatured hybrids. The dsRNA species migrated at higher molecular weight positions in the denaturing gels due to their synthesis by back-priming initiation. (C) The QDE-1 RdRP products with 0 or 70 nM RPA were treated with recombinant Dicer and resolved in 6% (top) or 16% (bottom) urea-containing polyacrylamide gels. The DNA/RNA hybrid refers to the single-stranded, labeled RNA products of the denatured hybrids.

Figure 4. The interaction between QDE-1 and RPA-1 requires QDE-3.

c-Myc-tagged QDE-3 and FLAG-tagged RPA-1 constructs were co-transformed into wild-type Neurospora (A and B) and qde-3^ko (B) strains. (A) Immunoprecipitation (IP) with a c-Myc specific antibody showing the interaction of c-Myc-QDE-3 with FLAG-RPA-1. The strain expressing only FLAG-RPA-1 was used as the control. (B) FLAG-IP showing the interaction between FLAG-QDE-1 and c-Myc-RPA-1, and the disappearance of this interaction in the qde-3^ko strain. The strain expressing only Myc-RPA-1 was used as the control. The antibodies used in the western analyses are indicated on the left. The strains and the fusion protein expressed are indicated below and above the panels, respectively.