Daniel Fisher

University of Texas Southwestern Medical Center, Dallas, TX, United States

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Publications (1)11.77 Total impact

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    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.
    PLoS Biology 10/2010; 8(10). DOI:10.1371/journal.pbio.1000496 · 11.77 Impact Factor