拟南芥中由特定 RNA 聚合酶产生的长非编码 RNA 的分析。
Analysis of long non-coding RNAs produced by a specialized RNA polymerase in Arabidopsis thaliana.
机构信息
University of Michigan, Department of Molecular, Cellular, and Developmental Biology, Ann Arbor, MI 48109, USA.
出版信息
Methods. 2013 Sep 15;63(2):160-9. doi: 10.1016/j.ymeth.2013.05.006. Epub 2013 May 22.
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.
长非编码 RNA（lncRNA）在包括基因表达调控在内的多个过程中发挥重要作用。在拟南芥中，一类 lncRNA 是由一种特殊的 RNA 聚合酶 V（Pol V）产生的，它通过转录基因沉默参与控制基因组活性。Pol V 产生的 lncRNA 被提议作为结合几个沉默因子的支架，这些因子进一步介导建立抑制性染色质修饰。我们提出了用于发现和表征由 Pol V 产生的 lncRNA 的方法。染色质免疫沉淀结合深度测序（ChIP-seq）允许以依赖于 Pol V 或由 Pol V 产生的转录物的方式发现与蛋白质结合的基因组区域。RNA 免疫沉淀（RIP）允许在鉴定的基因座上测试 lncRNA-蛋白相互作用。最后，实时 RT-PCR 允许从总 RNA 中检测低丰度的 Pol V 转录物。这些方法可能更广泛地应用于发现和表征由不同 RNA 聚合酶产生的 RNA。
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