Abstract
RNA-directed DNA methylation (RdDM) is a nuclear pathway which is comprised of multiple main and accessory protein components, including two plant-specific DNA-dependent RNA polymerases, Pol IV and Pol V, and argonaute (AGO) proteins. Regulation in the RdDM pathway can be achieved via multiple mechanisms, including the spatial distribution of different RdDM components. Here we describe a protocol for dissecting the subnuclear localization of AGO proteins and other RdDM components, including nuclei extraction from seedlings, slide preparation, and subsequent immunostaining.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10(2):94–108. doi:10.1038/nrg2504
Matzke MA, Mosher RA (2014) RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat Rev Genet 15(6):394–408. doi:10.1038/nrg3683
Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11(3):204–220. doi:10.1038/nrg2719
Zhang H, Zhu JK (2011) RNA-directed DNA methylation. Curr Opin Plant Biol 14(2):142–147. doi:10.1016/j.pbi.2011.02.003
Pikaard CS, Haag JR, Ream T, Wierzbicki AT (2008) Roles of RNA polymerase IV in gene silencing. Trends Plant Sci 13(7):390–397. doi:10.1016/j.tplants.2008.04.008
Wierzbicki AT, Haag JR, Pikaard CS (2008) Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135(4):635–648. doi:10.1016/j.cell.2008.09.035
Mallory A, Vaucheret H (2010) Form, function, and regulation of ARGONAUTE proteins. Plant Cell 22(12):3879–3889. doi:10.1105/tpc.110.080671
Havecker ER, Wallbridge LM, Hardcastle TJ, Bush MS, Kelly KA, Dunn RM, Schwach F, Doonan JH, Baulcombe DC (2010) The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22(2):321–334. doi:10.1105/tpc.109.072199
Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299(5607):716–719. doi:10.1126/science.1079695
Zheng X, Zhu J, Kapoor A, Zhu JK (2007) Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J 26(6):1691–1701. doi:10.1038/sj.emboj.7601603
Olmedo-Monfil V, Duran-Figueroa N, Arteaga-Vazquez M, Demesa-Arevalo E, Autran D, Grimanelli D, Slotkin RK, Martienssen RA, Vielle-Calzada JP (2010) Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464(7288):628–632. doi:10.1038/nature08828
Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, Jacobsen SE (2006) An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126(1):93–106. doi:10.1016/j.cell.2006.05.032
Pontes O, Li CF, Costa Nunes P, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS (2006) The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126(1):79–92. doi:10.1016/j.cell.2006.05.031
Acknowledgments
This work was supported by the Chinese Academy of Sciences and by National Institutes of Health Grant R01GM070795 (to J.–K. Z).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Duan, CG., Zhu, JK. (2017). Dissecting the Subnuclear Localization Patterns of Argonaute Proteins and Other Components of the RNA-Directed DNA Methylation Pathway in Plants. In: Carbonell, A. (eds) Plant Argonaute Proteins. Methods in Molecular Biology, vol 1640. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7165-7_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7165-7_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7164-0
Online ISBN: 978-1-4939-7165-7
eBook Packages: Springer Protocols