title: Isolation and profiling of protein-associated small RNAs.
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.
authors.affiliation: Department of Plant Pathology and Microbiology, Center for Plant Cell Biology and Institute for Integrative Genome Biology, University of California, Riverside, CA, USA.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Zhao
authors.forename: Yifan
authors.initials: Y
authors.lastname: Lii
authors.forename: Pei
authors.initials: P
authors.lastname: Zhu
authors.forename: Hailing
authors.initials: H
authors.lastname: Jin
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D029681:Arabidopsis Proteins / Q000378:metabolism; D060565:Argonaute Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006146:Guanidines / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing; D054884:Host-Pathogen Interactions; D007113:Immunity, Innate; D047468:Immunoprecipitation / Q000379:methods; D035683:MicroRNAs / Q000235:genetics / Q000302:isolation & purification*; D010636:Phenols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D044224:Pseudomonas syringae / Q000276:immunology / Q000502:physiology; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D058727:RNA, Small Untranslated / Q000235:genetics / Q000302:isolation & purification; D016601:RNA-Binding Proteins / Q000378:metabolism; D048348:Reverse Transcription; D017423:Sequence Analysis, RNA; D012997:Solvents / Q000737:chemistry
chemical_list: C117457:AGO1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D006146:Guanidines; C559857:MIRN393 microRNA, Arabidopsis; D035683:MicroRNAs; D010636:Phenols; D018749:RNA, Plant; D058727:RNA, Small Untranslated; D016601:RNA-Binding Proteins; D012997:Solvents; C559855:argonaute 2 protein, Arabidopsis; C411644:trizol