Browsing by Author "Mishra, Vibhor"

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    Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV
    (National Academy of Sciences, 2021-03-30) Mishra, Vibhor; Singh, Jasleen; Wang, Feng; Zhang, Yixiang; Fukudome, Akihito; Trinidad, Jonathan C.; Takagi, Yuichiro; Pikaard, Craig S.; Biochemistry and Molecular Biology, School of Medicine
    In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV's 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3' ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).
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    Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2
    (National Academy of Science, 2021) Fukudome, Akihito; Singh, Jasleen; Mishra, Vibhor; Reddem, Eswar; Martinez-Marquez, Francisco; Wenzel, Sabine; Yan, Rui; Shiozaki, Momoko; Yu, Zhiheng; Wang, Joseph Che-Yen; Takagi, Yuichiro; Pikaard, Craig S.; Biochemistry and Molecular Biology, School of Medicine
    RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.