[Show abstract][Hide abstract]ABSTRACT: Full-length, recombinant QDE-1 was used in the same RNA polymerase assay as described in Figure 1C, and shown are the ethidium bromide stained native agarose gels. Upper panel: ssRNA template; lower panel: ssDNA template. The activity of the full-length QDE-1 is identical to that of QDE-1 ΔN.
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[Show abstract][Hide abstract]ABSTRACT: (A) RNA polymerase reactions showing the association of QDE-1 with ssRNA and ssDNA in the presence and absence of NTP. 32P-labeled ssRNA (left panel) and ssDNA (right panel) templates were used. (B) RNA polymerase reactions using Myc-QDE-1 purified from Neurospora showing that QDE-1 cannot use dsDNA as a template.
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[Show abstract][Hide abstract]ABSTRACT: Coomassie-stained SDS-PAGE gels showing the purified truncated (above) and full-length QDE-1 (below). Recombinant QDE-1 proteins expressed in yeast were purified by a Ni-NTA column, a heparin column followed by an ion-exchange column. Protein fractions after the ion-exchange column are shown. The top fractions were pooled and concentrated before use in RNA polymerase assays. Full-length QDE-1 is ∼160 kDa and QDE-1 ΔN is ∼120 kDa in size.
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[Show abstract][Hide abstract]ABSTRACT: Author Summary
Small RNA molecules (20–30 nucleotides) play important roles in many cellular processes in eukaryotic organisms by silencing gene expression. To generate the many forms of small RNAs, DNA is first transcribed to produce single-stranded RNA (ssRNA), which then is converted to double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase (RdRP). However, it is not clear how the ssRNA templates are synthesized from DNA and specifically recognized by RdRPs amidst a sea of single-stranded, cellular RNAs. We previously showed that in the filamentous fungus Neurospora the production of one type of small RNA called qiRNA, which is specifically induced after DNA damage, requires the RdRP QDE-1. Here, we investigated the precise contributions of QDE-1 to the synthesis of ssRNA and dsRNA. We show that QDE-1 is surprisingly promiscuous in its template choice in that it is able to synthesize RNA from both ssRNA and single-stranded DNA (ssDNA). These results suggest that QDE-1 first generates ssRNA from a DNA template and then converts the ssRNA into dsRNA; this combination of activities in one protein ensures the specific action by RdRP on aberrant RNA in lieu of other single-stranded cellular RNA. In addition, we identified Replication Protein A, a ssDNA-binding protein that interacts with QDE-1, as an essential factor for small RNA production. Furthermore, we were able to reconstitute synthesis of dsRNA from ssDNA in a test tube using purified QDE-1 and RPA proteins, demonstrating the ability of this relatively simple biosynthetic system to generate the nucleic acid trigger for gene regulation. Together, these results uncover the details of a new and important small RNA production mechanism in cells.