SHH1, a Homeodomain Protein Required for DNA Methylation, As Well As RDR2, RDM4, and Chromatin Remodeling Factors, Associate with RNA Polymerase IV

SummaryIn eukaryotes with multiple small RNA pathways the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the siRNA biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV) whose atypical termination mechanism, induced by nontemplate DNA basepairing, channels transcripts to the associated RNA-dependent RNA polymerase, RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs then typically adds an extra untemplated 3’ terminal nucleotide to the second strands. The dicer endonuclease, DCL3 cuts resulting duplexes to generate 24 and 23nt siRNAs. The 23nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling and RNA channeling from template DNA transcription to siRNA guide strand/passenger strand discrimination.

Download Full-text

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

10.1101/2021.09.06.459145 ◽

2021 ◽

Author(s):

Andrew Loffer ◽

Jasleen Singh ◽

Akihito Fukudome ◽

Vibhor Mishra ◽

Feng Wang ◽

...

Keyword(s):

Dna Methylation ◽

Rna Polymerase ◽

Transferase Activity ◽

Rna Dependent Rna Polymerase ◽

Dna Viruses ◽

Selfish Genetic Elements ◽

Pol Iv ◽

Nuclear Rna ◽

Rna Polymerase Iv ◽

Nuclear Rna Polymerase

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.

Download Full-text

Plant Nuclear RNA Polymerase IV Mediates siRNA and DNA Methylation-Dependent Heterochromatin Formation

Cell ◽

10.1016/j.cell.2005.02.007 ◽

2005 ◽

Vol 120 (5) ◽

pp. 613-622 ◽

Cited By ~ 429

Author(s):

Yasuyuki Onodera ◽

Jeremy R. Haag ◽

Thomas Ream ◽

Pedro Costa Nunes ◽

Olga Pontes ◽

...

Keyword(s):

Dna Methylation ◽

Rna Polymerase ◽

Heterochromatin Formation ◽

Nuclear Rna ◽

Rna Polymerase Iv ◽

Nuclear Rna Polymerase

Download Full-text

Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1413053112 ◽

2015 ◽

Vol 112 (3) ◽

pp. 917-922 ◽

Cited By ~ 71

Author(s):

Donna M. Bond ◽

David C. Baulcombe

Keyword(s):

Dna Methylation ◽

Rna Polymerase ◽

Gene Silencing ◽

De Novo ◽

Feedback Mechanism ◽

Epigenetic Silencing ◽

Virus Induced Gene Silencing ◽

Epigenetic Modifiers ◽

Rna Polymerase Iv ◽

Genetic Mutants

In plants, RNA-directed DNA methylation (RdDM), a mechanism where epigenetic modifiers are guided to target loci by small RNAs, plays a major role in silencing of transposable elements (TEs) to maintain genome integrity. So far, two RdDM pathways have been identified: RNA Polymerase IV (PolIV)-RdDM and RNA-dependent RNA Polymerase 6 (RDR6)-RdDM. PolIV-RdDM involves a self-reinforcing feedback mechanism that maintains TE silencing, but cannot explain how epigenetic silencing is first initiated. A function of RDR6-RdDM is to reestablish epigenetic silencing of active TEs, but it is unknown if this pathway can induce DNA methylation at naïve, non-TE loci. To investigate de novo establishment of RdDM, we have used virus-induced gene silencing (VIGS) of an active FLOWERING WAGENINGEN epiallele. Using genetic mutants we show that unlike PolIV-RdDM, but like RDR6-RdDM, establishment of VIGS-mediated RdDM requires PolV and DRM2 but not Dicer like-3 and other PolIV pathway components. DNA methylation in VIGS is likely initiated by a process guided by virus-derived small (s) RNAs that are 21/22-nt in length and reinforced or maintained by 24-nt sRNAs. We demonstrate that VIGS-RdDM as a tool for gene silencing can be enhanced by use of mutant plants with increased production of 24-nt sRNAs to reinforce the level of RdDM.

Download Full-text

Faculty Opinions recommendation of Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1024633.290785 ◽

2005 ◽

Author(s):

Sui Huang

Keyword(s):

Dna Methylation ◽

Rna Polymerase ◽

Heterochromatin Formation ◽

Nuclear Rna ◽

Rna Polymerase Iv ◽

Nuclear Rna Polymerase

Download Full-text

Arabidopsis RNA Polymerase IV generates 21-22 nucleotide small RNAs that can participate in RNA-directed DNA methylation and may regulate genes

10.1101/832634 ◽

2019 ◽

Author(s):

Kaushik Panda ◽

Andrea D. McCue ◽

R. Keith Slotkin

Keyword(s):

Dna Methylation ◽

Transposable Elements ◽

Rna Polymerase ◽

Transcript Abundance ◽

Gene Transcript ◽

Small Interfering Rnas ◽

Pol Iv ◽

Rna Polymerase Iv ◽

Plant Life Cycle ◽

And Function

AbstractThe plant-specific RNA Polymerase IV (Pol IV) transcribes heterochromatic regions, including many transposable elements, with the well-described role of generating 24 nucleotide (nt) small interfering RNAs (siRNAs). These siRNAs target DNA methylation back to transposable elements to reinforce the boundary between heterochromatin and euchromatin. In the male gametophytic phase of the plant life cycle, pollen, Pol IV switches to generating primarily 21-22 nt siRNAs, but the biogenesis and function of these siRNAs has been enigmatic. In contrast to being pollen-specific, we identified that Pol IV generates these 21-22 nt siRNAs in sporophytic tissues, likely from the same transcripts that are processed into the more abundant 24 nt siRNAs. The 21-22 nt forms are specifically generated by the combined activities of DICER proteins DCL2/DCL4 and can participate in RNA-directed DNA methylation. These 21-22 nt siRNAs are also loaded into ARGONAUTE1, which is known to function in post-transcriptional regulation. Like other plant siRNAs and microRNAs incorporated into AGO1, we find a signature of genic mRNA cleavage at the predicted target site of these siRNAs, suggesting that Pol IV-generated 21-22 nt siRNAs may function to regulate gene transcript abundance. Our data provides support for the existing model that in pollen Pol IV functions in gene regulation.

Download Full-text

Epitope selection for multipeptide proteins: the case of RNA polymerase IV and V

Biologia ◽

10.2478/s11756-012-0085-y ◽

2012 ◽

Vol 67 (5) ◽

Author(s):

Leydi Canche Moo ◽

Analy Arroyo Herrera ◽

Luis Rodriguez-Zapata ◽

Victor Suarez ◽

Enrique Castano

Keyword(s):

Dna Methylation ◽

Rna Polymerase ◽

Protein Complexes ◽

Selection Process ◽

Viral Control ◽

Rna Polymerase Iv ◽

Dna Silencing ◽

Interfering Rna ◽

Epitope Selection ◽

Rna And Dna

AbstractLarge multipeptide protein complexes have provided a challenge for epitope selection, which is required for immunological protocols where native conformations are needed. Immunolocalization requires native conformation of the proteins, which is essential for further understanding of biological activity. RNA polymerase IV and V are multisubunit proteins that interact with other factors in the RNA-directed DNA methylation pathway for control of DNA silencing by small interfering RNA and DNA methylation. DNA silencing is an important process during cell differentiation, nuclear structure and viral control among others. RNA polymerase IV and V are yet to be studied in model monocot systems like Oryza sativa, which may provide further data to define if the genetic silencing mechanism has diverged over time as compared to dicots. Here we show an in silico selection process of exposed sequences and their use for obtaining antibodies against native RNA polymerase IV and V in O. sativa.

Download Full-text