Abstract
Small RNAs are short noncoding RNAs with important regulatory roles in many cellular processes. Small RNAs are generated by DICER or DICER-like proteins and then incorporated into RNAi effector proteins ARGONAUTEs (AGOs) for silencing of their targets. In plants, small RNAs regulate host innate immunity against various pathogens, but their mode of action and associated protein factors that facilitate their function remain to be elucidated. Here, we describe an efficient method to isolate AGO-associated small RNAs from Arabidopsis. This protocol can be easily adapted for the isolation of any protein-associated small RNAs. We utilized immunoprecipitation tandem with deep sequencing to identify small RNAs with functions in plant innate immunity. Using this described protocol, we identified miR393* that plays a crucial role in plant antibacterial defense. The distinct roles played by individual AGO proteins were observed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10(2):94–108
Mallory AC, Vaucheret H (2006) Functions of microRNAs and related small RNAs in plants. Nat Genet 38:S31–S36
Inui M, Martello G, Piccolo S (2010) MicroRNA control of signal transduction. Nat Rev Mol Cell Biol 11(4):252–263
van Rooij E et al (2007) Control of stress-dependent cardiac growth and gene expression by a MicroRNA. Science 316(5824):575–579
Voinnet O (2009) Origin, biogenesis, and activity of plant MicroRNAs. Cell 136(4):669–687
Vaucheret H (2008) Plant ARGONAUTES. Trends Plant Sci 13(7):350–358
Zhang XM et al (2011) Arabidopsis argonaute 2 regulates innate immunity via miRNA393*-mediated silencing of a golgi-localized SNARE gene, MEMB12. Mol Cell 42(3):356–366
Harvey JJ et al (2011) An antiviral defense role of AGO2 in plants. PLoS One 6(1):e14639
Mi S et al (2008) Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 52 terminal nucleotide. Cell 133(1):116–127
Montgomery TA et al (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133(1):128–141
Katiyar-Agarwal S, Jin HL (2007) Discovery of pathogen-regulated small RNAs in plants. In: Rossi JJ, Hannon GJ (eds) Academic Press; San Diego, California. Methods Enzymol 427:215–227
Sambrook, J. (2001) Molecular cloning: a laboratory manual Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press
Acknowledgments
We thank Jim Carrington for providing the HA:GUS and pAGO2:HA-AGO2 lines. The research work is supported by an NIH R01 GM093008, an NSF Career Award MCB-0642843, and an AES-CE Research Allocation Award PPA-7517H to H. Jin.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zhao, H., Lii, Y., Zhu, P., Jin, H. (2012). Isolation and Profiling of Protein-Associated Small RNAs. In: Jin, H., Gassmann, W. (eds) RNA Abundance Analysis. Methods in Molecular Biology, vol 883. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-839-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-839-9_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-838-2
Online ISBN: 978-1-61779-839-9
eBook Packages: Springer Protocols