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

  1. Todd Blevins
  2. Ram Podicheti
  3. Vibhor Mishra
  4. Michelle Marasco
  5. Jing Wang
  6. Douglas Rusch
  7. Haixu Tang
  8. Craig S Pikaard  Is a corresponding author
  1. Université de Strasbourg, France
  2. Indiana University, United States
  3. Howard Hughes Medical Institute, Indiana University, United States

Abstract

In Arabidopsis thaliana, abundant 24 nt siRNAs 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-LIKE3 (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. 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.

Article and author information

Author details

  1. Todd Blevins

    : Institut de Biologie Moléculaire des Plantes du CNRS, UPR2357, Université de Strasbourg, Strasbourg, France
    Competing interests
    The authors declare that no competing interests exist.
  2. Ram Podicheti

    Center for Genomics and Bioinformatics, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Vibhor Mishra

    Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Michelle Marasco

    Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Jing Wang

    Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Douglas Rusch

    Center for Genomics and Bioinformatics, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Haixu Tang

    School of Informatics and Computing, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Craig S Pikaard

    Howard Hughes Medical Institute, Indiana University, Bloomington, United States
    For correspondence
    cpikaard@indiana.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Daniel Zilberman, University of California, Berkeley, United States

Version history

  1. Received: June 21, 2015
  2. Accepted: October 1, 2015
  3. Accepted Manuscript published: October 2, 2015 (version 1)
  4. Version of Record published: December 8, 2015 (version 2)

Copyright

© 2015, Blevins et al.

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

Metrics

  • 4,119
    views
  • 1,160
    downloads
  • 204
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Todd Blevins
  2. Ram Podicheti
  3. Vibhor Mishra
  4. Michelle Marasco
  5. Jing Wang
  6. Douglas Rusch
  7. Haixu Tang
  8. Craig S Pikaard
(2015)
Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis
eLife 4:e09591.
https://doi.org/10.7554/eLife.09591

Share this article

https://doi.org/10.7554/eLife.09591

Further reading

    1. Chromosomes and Gene Expression
    2. Immunology and Inflammation
    Rajan M Thomas, Matthew C Pahl ... Andrew D Wells
    Research Article

    Ikaros is a transcriptional factor required for conventional T cell development, differentiation, and anergy. While the related factors Helios and Eos have defined roles in regulatory T cells (Treg), a role for Ikaros has not been established. To determine the function of Ikaros in the Treg lineage, we generated mice with Treg-specific deletion of the Ikaros gene (Ikzf1). We find that Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibit Th1-like gene expression with abnormal production of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signaling. While Ikzf1-Treg-cko mice do not develop spontaneous autoimmunity, Ikaros-deficient Treg are unable to control conventional T cell-mediated immune pathology in response to TCR and inflammatory stimuli in models of IBD and organ transplantation. These studies establish Ikaros as a core factor required in Treg for tolerance and the control of inflammatory immune responses.

    1. Cell Biology
    2. Chromosomes and Gene Expression
    Lucie Crhak Khaitova, Pavlina Mikulkova ... Karel Riha
    Research Article

    Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.