A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation

In plants, selfish genetic elements including retrotransposons and DNA viruses are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of ~30 bp synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV initiating nucleotide choice, RDR2 initiation 1-2 nt internal to Pol IV transcript ends and RDR2 terminal transferase activity collectively yield a code that influences which end of the precursor is diced and whether 24 or 23 nt siRNAs are generated from the Pol IV or RDR2-transcribed strands. By diversifying the size, sequence, and strand polarity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow maximal siRNA coverage at methylated target loci.

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Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019276118 ◽

2021 ◽

Vol 118 (13) ◽

pp. e2019276118

Author(s):

Vibhor Mishra ◽

Jasleen Singh ◽

Feng Wang ◽

Yixiang Zhang ◽

Akihito Fukudome ◽

...

Keyword(s):

Rna Polymerase ◽

Active Site ◽

Rna Dependent Rna Polymerase ◽

Dna Viruses ◽

Selfish Genetic Elements ◽

Pol Iv ◽

Nuclear Rna ◽

Tightly Coupled ◽

Rna Polymerase Iv ◽

Nuclear Rna Polymerase

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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Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

eLife ◽

10.7554/elife.09591 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 120

Author(s):

Todd Blevins ◽

Ram Podicheti ◽

Vibhor Mishra ◽

Michelle Marasco ◽

Jing Wang ◽

...

Keyword(s):

Rna Polymerase ◽

Cytosine Methylation ◽

De Novo ◽

Transferase Activity ◽

Small Interfering Rnas ◽

Pol Iv ◽

Nuclear Rna ◽

Rna Polymerase Iv ◽

Nuclear Rna Polymerase

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

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Assembly of a double-stranded RNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 docks with NUCLEAR RNA POLYMERASE IV at the clamp domain

10.1101/2020.09.09.290171 ◽

2020 ◽

Author(s):

Vibhor Mishra ◽

Jasleen Singh ◽

Akihito Fukudome ◽

Feng Wang ◽

Yixiang Zhang ◽

...

Keyword(s):

Rna Polymerase ◽

Mrna Translation ◽

Transcriptional Gene Silencing ◽

Exit Channel ◽

Rna Dependent Rna Polymerase ◽

Pol Iv ◽

Nuclear Rna ◽

Rna Polymerase Iv ◽

Rapid Conversion ◽

Nuclear Rna Polymerase

AbstractIn 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 co-immunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that RDR2 stably associates with Pol IV’s largest catalytic subunit, NRPD1 at three sites, all within the clamp module. The clamp is a ubiquitous feature of DNA-dependent RNA polymerases that opens to allow DNA template entry and closes to encase the DNA-RNA hybrid adjacent to the RNA exit channel. The clamp also provides binding sites for polymerase-specific subunits or regulatory proteins, thus RDR2 binding to the Pol IV clamp is consistent with this theme. Within RDR2, the site of interaction with NRPD1 is very near the catalytic center. The locations of the NRPD1-RDR2 contact sites suggest a model in which transcripts emanating from Pol IV’s RNA exit channel align with the template cleft of RDR2, facilitating rapid conversion of terminated Pol IV transcripts into double-stranded RNAs.Significance StatementShort interfering RNAs (siRNAs) play important roles in gene regulation by inhibiting mRNA translation into proteins or by guiding chromatin modifications that inhibit gene transcription. In plants, transcriptional gene silencing is guided by siRNAs derived from double-stranded (ds) RNAs generated by coupling the activities of DNA-dependent NUCLEAR RNA POLYMERASE IV and RNA-DEPENDENT RNA POLYMERASE 2. We show that the physical basis for Pol IV-RDR2 coupling is RDR2 binding to the clamp domain of Pol IV’s largest subunit. The positions of the protein docking sites suggest that nascent Pol IV transcripts are generated in close proximity to RDR2’s catalytic site, enabling rapid conversion of Pol IV transcripts into dsRNAs.

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