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

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Abstract

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 that are synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV’s choice of initiating nucleotide, RDR2’s initiation 1–2 nt internal to Pol IV transcript ends and RDR2’s terminal transferase activity collectively yield a code that influences which precursor end is diced and whether 24 or 23 nt siRNAs are produced. By diversifying the size, sequence, and strand specificity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow for maximal siRNA coverage at methylated target loci.

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RNA-directed DNA methylation: an epigenetic pathway of increasing complexity.

Marjori Matzke, Rebecca A. Mosher (2014)

RNA-directed DNA methylation (RdDM) is the major small RNA-mediated epigenetic pathway in plants. RdDM requires a specialized transcriptional machinery that comprises two plant-specific RNA polymerases - Pol IV and Pol V - and a growing number of accessory proteins, the functions of which in the RdDM mechanism are only partially understood. Recent work has revealed variations in the canonical RdDM pathway and identified factors that recruit Pol IV and Pol V to specific target sequences. RdDM, which transcriptionally represses a subset of transposons and genes, is implicated in pathogen defence, stress responses and reproduction, as well as in interallelic and intercellular communication.

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Single processing center models for human Dicer and bacterial RNase III.

Haidi Zhang, Fabrice Kolb, Lukasz Jaskiewicz … (2004)

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. We have generated mutations in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and studied their effect on RNA processing. Our results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, we propose that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.

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Dicer recognizes the 5' end of RNA for efficient and accurate processing.

Jong-Eun Park, Inha Heo, Yuan Tian … (2011)

A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3' overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3' end but also the 5' end, with the cleavage site determined mainly by the distance (∼22 nucleotides) from the 5' end (5' counting rule). This cleavage requires a 5'-terminal phosphate group. Further, we identify a novel basic motif (5' pocket) in human Dicer that recognizes the 5'-phosphorylated end. The 5' counting rule and the 5' anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5' pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5'-pocket mutant. Thus, 5'-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs. ©2011 Macmillan Publishers Limited. All rights reserved

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Author and article information

Contributors

Craig S Pikaard:

ORCID: https://orcid.org/0000-0001-8204-7459

Daniel Zilberman: Role: Reviewing Editor

James L Manley: Role: Senior Editor

Journal

Journal ID (nlm-ta): eLife

Journal ID (iso-abbrev): Elife

Journal ID (publisher-id): eLife

Title: eLife

Publisher: eLife Sciences Publications, Ltd

ISSN (Electronic): 2050-084X

Publication date (Electronic, pub): 31 January 2022

Publication date Collection: 2022

Volume: 11

Electronic Location Identifier: e73260

Affiliations

[1 ] Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University Bloomington ( https://ror.org/02k40bc56) Bloomington United States

[2 ] Howard Hughes Medical Institute, Indiana University ( https://ror.org/01kg8sb98) Bloomington United States

Institue of Science and Technology ( https://ror.org/03gnh5541) Austria

Columbia University ( https://ror.org/00hj8s172) United States

Institue of Science and Technology ( https://ror.org/03gnh5541) Austria

Tokyo University of Agriculture and Technology ( https://ror.org/00qg0kr10) Japan

Author information

Akihito Fukudome https://orcid.org/0000-0001-8924-1035

Craig S Pikaard https://orcid.org/0000-0001-8204-7459

Article

Publisher ID: 73260

DOI: 10.7554/eLife.73260

PMC ID: 8846587

PubMed ID: 35098919

SO-VID: be2576ee-521e-4dc6-81b7-e2710bc98a26

License:

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

History

Date received : 23 August 2021

Date accepted : 28 January 2022

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: GM077590

Award Recipient : Craig S Pikaard

Funded by: FundRef http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;

Award ID: Investigator:Pikaard

Award Recipient : Akihito Fukudome Vibhor Mishra Feng Wang Craig S Pikaard

Funded by: FundRef http://dx.doi.org/10.13039/100006733, Indiana University;

Award ID: Carlos O. Miller Graduate Student Fellowship

Award Recipient : Andrew Loffer Jasleen Singh

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Author impact statement Double-stranded precursors of siRNAs involved in RNA-directed DNA methylation have initiating sequences and overhangs imparted by the RNA polymerases that synthesize them, programming alternative dicing reactions that diversify the siRNA pool.

ScienceOpen disciplines: Life sciences

Keywords: dicer endonuclease,dicer-like 3,sirna biogenesis,rna-directed dna methylation,rna silencing,rna polymerase,a. thaliana

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ScienceOpen disciplines: Life sciences

Keywords: dicer endonuclease, dicer-like 3, sirna biogenesis, rna-directed dna methylation, rna silencing, rna polymerase, a. thaliana

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

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Abstract

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Most cited references 41

RNA-directed DNA methylation: an epigenetic pathway of increasing complexity.

Single processing center models for human Dicer and bacterial RNase III.

Dicer recognizes the 5' end of RNA for efficient and accurate processing.

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