Ream, T. al. Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II. Mol. Cell33, 192-203

Biology Department, Washington University, St. Louis, MO 63130, USA.
Molecular cell (Impact Factor: 14.02). 01/2009; 33(2):192-203. DOI: 10.1016/j.molcel.2008.12.015
Source: PubMed


In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.

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    • "This pattern is very similar to that known for the transcriptional start sites (TSSs) of RNA Polymerase II (Pol II) in plants and other organisms and it is referred to as the ''Y/R rule'' (Cumbie et al., 2015; Nechaev et al., 2010; Yamamoto et al., 2007). This result suggests that Pol IV has retained this preference from its evolutionary ancestor Pol II (Ream et al., 2009), and that the 5 0 ends of P4RNAs likely represent Pol IV transcriptional start sites. The short TSS like sequences at the 5 0 ends of P4RNA, along with their very short nature does not support previous models in which Pol IV initiates transcription solely at the nucleosomedepleted promoter regions near the ends of transposons to produce long transcripts (Li et al., 2015). "
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    ABSTRACT: RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5'-adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured with 5'-monophosphates and 3'-misincorporated nucleotides. The 3'-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a "one precursor, one siRNA" model for the biogenesis of 24-nt siRNAs in Arabidopsis.
    Cell 10/2015; 163(2):p445-455. DOI:10.1016/j.cell.2015.09.032 · 32.24 Impact Factor
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    • "We also examined the transcript levels of genes involved in two other sRNA pathways. They include SGS3, RDR6, DRB4, and DCL4 of the ta-siRNA pathway and NRPD1a (encodes the largest subunit of Pol IV) (Ream et al., 2009), RDR2, DCL3, NRPE1 (encodes the largest subunit of Pol V) (Ream et al., 2009), AGO4, and DRM2 of the hc-siRNA pathway. We found that the transcript levels of DCL4 and DCL3 were reduced by 50%– 60%, while the expression of other genes was unchanged in xct-2 relative to Col-0 (Figure 5D and 5E). "
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    ABSTRACT: Small RNAs (sRNAs) play important regulatory roles in various aspects of plant biology. They are processed from double-stranded RNA precursors by Dicer-like (DCL) proteins. There are three major classes of sRNAs in Arabidopsis: DCL1-dependent microRNA (miRNA), DCL3-dependent heterochromatic siRNA (hc-siRNA) and DCL4-dependent trans-acting siRNA (ta-siRNA). We have previously isolated a mutant with compromised miRNA activity, cma33. Here we show CMA33 encodes a nuclear localized protein XCT (XAP5 CIRCADIAN TIMEKEEPER). The cma33/xct mutation led to reduced accumulation of not only miRNAs but also hc-siRNAs and ta-siRNAs. Intriguingly, we found that the expression of DCL1, DCL3 and DCL4, but not other genes in the sRNA biogenesis pathways, was decreased in cma33/xct. Consistent with this, the occupancy of Pol II at DCL1, DCL3 and DCL4 genes was reduced upon the loss of CMA33/XCT. Collectively, our data suggest that CMA33/XCT modulates sRNA production through regulating the transcription of DCLs. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.
    Molecular Plant 03/2015; 8(8). DOI:10.1016/j.molp.2015.03.002 · 6.34 Impact Factor
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    • "Pol IV is a Pol II-derived plant specific polymerase. It contains many identical subunits of Pol II [22], but the largest subunit NRPD1 and the second largest subunit NRPD2/NRPE2 of pol IV are paralogous of their counterparts in Pol II [22]. Over 90% siRNAs require Pol IV for their production [23]. "
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    ABSTRACT: RNA silencing is a process triggered by 21-24 small RNAs to repress gene expression. Many organisms including plants use RNA silencing to regulate development and physiology, and to maintain genome stability. Plants possess two classes of small RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The frameworks of miRNA and siRNA pathways have been established in the model plant, Arabidopsis thaliana (Arabidopsis). Here we report the identification of putative genes that are required for the generation and function of miRNAs and siRNAs in soybean and sorghum, based on knowledge obtained from Arabidopsis. The gene families, including DCL, HEN1, SE, HYL1, HST, RDR, NRPD1, NRPD2/NRPE2, NRPE1, and AGO, were analyzed for gene structures, phylogenetic relationships, and protein motifs. The gene expression was validated using RNA-seq, expressed sequence tags (EST), and reverse transcription PCR (RT-PCR). The identification of these components could provide not only insight into RNA silencing mechanism in soybean and sorghum but also basis for further investigation. All data are available at
    BMC Bioinformatics 01/2014; 15(1):4. DOI:10.1186/1471-2105-15-4 · 2.58 Impact Factor
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