title: Analysis of long non-coding RNAs produced by a specialized RNA polymerase in Arabidopsis thaliana.
abstract: Long non-coding RNAs (lncRNAs) play important roles in several processes including control of gene expression. In Arabidopsis thaliana, a class of lncRNAs is produced by a specialized RNA Polymerase V (Pol V), which is involved in controlling genome activity by transcriptional gene silencing. lncRNAs produced by Pol V have been proposed to serve as scaffolds for binding of several silencing factors which further mediate the establishment of repressive chromatin modifications. We present methods for discovery and characterization of lncRNAs produced by Pol V. Chromatin Immunoprecipitation coupled with deep sequencing (ChIP-seq) allows discovery of genomic regions bound by proteins in a manner dependent on either Pol V or transcripts produced by Pol V. RNA Immunoprecipitation (RIP) allows testing lncRNA-protein interactions at identified loci. Finally, real-time RT-PCR allows detection of low abundance Pol V transcripts from total RNA. These methods may be more broadly applied to discovery and characterization of RNAs produced by distinct RNA Polymerases.
authors.affiliation: University of Michigan, Department of Molecular, Cellular, and Developmental Biology, Ann Arbor, MI 48109, USA.
authors.forename: M Jordan
authors.initials: MJ
authors.lastname: Rowley
authors.forename: Gudrun
authors.initials: G
authors.lastname: Böhmdorfer
authors.forename: Andrzej T
authors.initials: AT
authors.lastname: Wierzbicki
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000502:physiology*; D002843:Chromatin / Q000302:isolation & purification; D047369:Chromatin Immunoprecipitation; D018744:DNA, Plant / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D059014:High-Throughput Nucleotide Sequencing; D062085:RNA, Long Noncoding / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D018749:RNA, Plant / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D029681:Arabidopsis Proteins; D002843:Chromatin; D018744:DNA, Plant; D062085:RNA, Long Noncoding; D018749:RNA, Plant; C538841:RNA polymerase V, Arabidopsis; D012321:DNA-Directed RNA Polymerases
keywords: ChIP; Gene silencing; Non-coding RNA; RIP; RNA Polymerase