Reaction Mechanisms of Pol IV, RDR2, and DCL3 Drive RNA Channeling in the siRNA-Directed DNA Methylation Pathway

Singh, Jasleen; Mishra, Vibhor; Wang, Feng; Huang, Hsiao-Yun; Pikaard, Craig S.

doi:10.1016/j.molcel.2019.07.008

Cited by 96 publications

(158 citation statements)

References 56 publications

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“…A plant-specific RNA polymerase complex, RNA Polymerase IV (Pol IV), is first recruited to silent heterochromatin via its interaction with CLASSY (CLSY) proteins and SAWADEE homeodomain homolog 1 (SHH1) (also see 'Interactions between RdDM and other chromatin modifying pathways' below) [79][80][81]. Pol IV transcribes these regions to produce short single-stranded RNAs (ssRNAs) roughly 30 to 45 nucleotides in length, each of which is the precursor for a single sRNA [82][83][84]. These ssRNAs are converted into doublestranded RNAs (dsRNAs) co-transcriptionally by RNA-directed RNA polymerase 2 (RDR2), which physically associates with Pol IV [83].…”

Section: Fig 3 Schematic Of the Canonical Rddm Pathway (Top) And Nomentioning

confidence: 99%

“…The dsRNAs are then cleaved by the endoribonuclease Dicer-like 3 (DCL3) into 24 nucleotide (nt) sRNAs. Pol IV, RDR2, and DCL3 alone are sufficient for the production of 24 nt sRNAs in vitro [84], suggesting that while other factors involved in this part of the pathway may help increase efficiency or specificity, they are not required for Pol IV-mediated sRNA production.…”

Section: Fig 3 Schematic Of the Canonical Rddm Pathway (Top) And Nomentioning

confidence: 99%

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RNA-directed DNA Methylation

2020

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RNA-directed DNA methylation (RdDM) is a biological process in which non-coding RNA molecules direct the addition of DNA methylation to specific DNA sequences. The RdDM pathway is unique to plants, although other mechanisms of RNA-directed chromatin modification have also been described in fungi and animals. To date, the RdDM pathway is best characterized within angiosperms (flowering plants), and particularly within the model plant Arabidopsis thaliana. However, conserved RdDM pathway components and associated small RNAs (sRNAs) have also been found in other groups of plants, such as gymnosperms and ferns. The RdDM pathway closely resembles other sRNA pathways, particularly the highly conserved RNAi pathway found in fungi, plants, and animals. Both the RdDM and RNAi pathways produce sRNAs and involve conserved Argonaute, Dicer and RNAdependent RNA polymerase proteins. RdDM has been implicated in a number of regulatory processes in plants. The DNA methylation added by RdDM is generally associated with transcriptional repression of the genetic sequences targeted by the pathway. Since DNA methylation patterns in plants are heritable, these changes can often be stably transmitted to progeny. As a result, one prominent role of RdDM is the stable, transgenerational suppression of transposable element (TE) activity. RdDM has also been linked to pathogen defense, abiotic stress responses, and the regulation of several key developmental transitions. Although the RdDM pathway has a number of important functions, RdDM-defective mutants in Arabidopsis thaliana are viable and can reproduce, which has enabled detailed genetic studies of the pathway. However, RdDM mutants can have a range of defects in different plant species, including lethality, altered reproductive phenotypes, TE upregulation and genome instability, and increased pathogen sensitivity. Overall, RdDM is an important pathway in plants that regulates a number of processes by establishing and reinforcing specific DNA methylation patterns, which can lead to transgenerational epigenetic effects on gene expression and phenotype. Biological functions of RdDM RdDM is involved in a number of biological processes in the plant, including stress responses, cell-to-cell communication, and the maintenance of genome stability through TE silencing. An overview of some of the biological functions performed by RdDM is shown in Fig 1.

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Section: Fig 3 Schematic Of the Canonical Rddm Pathway (Top) And Nomentioning

confidence: 99%

Section: Fig 3 Schematic Of the Canonical Rddm Pathway (Top) And Nomentioning

confidence: 99%

RNA-directed DNA Methylation

2020

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“…RNA Pol IV and RDR2 together produce short double-stranded transcripts (Blevins et al, 2015;Zhai et al, 2015). Pol IV/RDR2 products are then cleaved by DICER-LIKE 3 (DCL3) to produce 24-nt siRNA duplexes (Singh et al, 2019) that can be bound by ARGONAUTE 4 (AGO4), which removes the "passenger strand," leaving the "guide strand" bound (Havecker et al, 2010;Wang and Axtell, 2017). The AGO4-siRNA complex interacts with the Cterminal domain of RNA Pol V and either the Pol V transcript or DNA within the Pol V transcription bubble (El-shami et al, 2007;Lahmy et al, 2016;Li et al, 2006;Liu et al, 2018), leading to the recruitment of the DNA methyltransferase DRM2 and methylation of cytosines (Matzke and Mosher, 2014).…”

Section: Introductionmentioning

confidence: 99%

Abundant expression of maternal siRNAs is a conserved feature of seed development

Grover

Burgess

Kendall

et al. 2019

Preprint

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Small RNAs are abundant in plant reproductive tissues, especially 24-nt short interfering (si)RNAs. Most 24-nt siRNAs are dependent on RNA Pol IV and RDR2, and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.

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“…Pol IV transcription is relatively error-prone compared to Pol II and Pol V, but there was no difference in nucleotide misincorporation at methylcytosines versus cytosines (Marasco et al 2017). Cytosine methylation also does not induce Pol IV termination in vitro (Marasco et al 2017;Singh et al 2019).…”

Section: In Vivo Studies and Unanswered Questionsmentioning

confidence: 92%

Reconstitution of siRNA Biogenesis In Vitro: Novel Reaction Mechanisms and RNA Channeling in the RNA-Directed DNA Methylation Pathway

Singh¹,

Pikaard

2019

Cold Spring Harb Symp Quant Biol

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Eukaryotes deploy RNA-mediated gene silencing pathways to guard their genomes against selfish genetic elements, such as transposable elements and invading viruses. In plants, RNA-directed DNA methylation (RdDM) is used to silence selfish elements at the level of transcription. This process involves 24-nt short interfering RNAs (siRNAs) and longer noncoding RNAs to which the siRNAs base-pair. Recently, we showed that 24-nt siRNA biogenesis could be recapitulated in the test tube using purified enzymes, yielding biochemical answers to numerous questions left unresolved by prior genetic and genomic studies. Interestingly, each enzyme has activities that program what happens in the next step, thus channeling the RNAs within the RdDM pathway and restricting their diversion into alternative pathways. However, a similar mechanistic understanding is lacking for other important steps of the RdDM pathway. We discuss some of the steps most in need of biochemical investigation and important questions still in need of answers.

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Reaction Mechanisms of Pol IV, RDR2, and DCL3 Drive RNA Channeling in the siRNA-Directed DNA Methylation Pathway

Cited by 96 publications

References 56 publications

RNA-directed DNA Methylation

RNA-directed DNA Methylation

Abundant expression of maternal siRNAs is a conserved feature of seed development

Reconstitution of siRNA Biogenesis In Vitro: Novel Reaction Mechanisms and RNA Channeling in the RNA-Directed DNA Methylation Pathway

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