Analysis of long non-coding RNAs produced by a specialized RNA polymerase in Arabidopsis thaliana
Introduction
Long non-coding RNA (lncRNA), among other functions, is involved in directing chromatin modifications in order to control genes and transposons [1]. These modifications include de novo DNA methylation, histone modifications, and nucleosome positioning in a pathway known as RNA-mediated transcriptional gene silencing or RNA-dependent DNA methylation (RdDM) [2], [3], [4]. Mutations in components of the RdDM pathway can cause increased transposon activity, faulty DNA repair, or misregulation of genes [4], [5], [6].
In Arabidopsis thaliana, a class of lncRNAs involved in RdDM is produced by RNA Polymerase V, a specialized DNA dependent RNA Polymerase [7]. Pol V transcripts are thought to create scaffolds on which several factors bind in order to control chromatin states [3], [8]. One of these factors is ARGONAUTE4 (AGO4), which binds to the Pol V C-terminal domain as well as to RNA and DNA [9], [10]. AGO4 is guided to chromatin by not only Pol V and its transcripts, but also by 24 nucleotide small interfering RNA (siRNA) [4], [8]. These siRNAs are generated from cleavage of double stranded transcripts produced by the coordinated activity of RNA Polymerase IV and RNA-dependent RNA Polymerase 2 [4], [11]. AGO4 associates with siRNA and is guided to chromatin based on the sequence specificity of the siRNA and the localization of Pol V [3], [5].
Another protein that binds to chromatin dependent on Pol V-produced lncRNA is SPT5-like (SPT5L) [12], [13], [14]. SPT5L works with AGO4 in a locus specific manner to direct the activity of the de novo DNA methyltransferase, DRM2 [2], [13].
Although DNA methylation is a major repressive chromatin modification established in RdDM, it is not the only one. Our recent work implicated Pol V-produced lncRNA in nucleosome positioning by indirectly recruiting a SWI/SNF chromatin remodeling complex via the IDN2 protein [15]. Being involved in the establishment of DNA methylation and repressive histone modifications as well as changes in nucleosome positioning, Pol V-produced lncRNA has broad effects on chromatin status and has been proposed to control genome activity [3].
Discovery of loci under control of Pol V-produced lncRNAs relies on finding genomic regions bound by Pol V [16], [17]. An additional approach is identifying binding sites of proteins recruited by Pol V transcripts [5]. Further investigation of the functional significance of those lncRNAs requires the ability to directly detect their presence and study their interactions with proteins [7], [9], [15], [16], [17]. We present methods, which allow the study of Pol V produced lncRNA as well as transcripts produced by other RNA Polymerases. Although methods we show have only been tested with plant tissue, they should be applicable to the wide array of eukaryotic organisms.
Section snippets
Chromatin immunoprecipitation (ChIP)
One of the main mechanisms used by lncRNA to control genome activity is by guiding proteins to specific genomic loci [3]. Detecting where these proteins are bound can give insights into the function of lncRNA [9], [13]. This approach may also be used in conjunction with high-throughput sequencing in order to discover new loci that are impacted by lncRNA [5], [16], [17]. Moreover, when performed in different mutant backgrounds, protein–DNA interaction assays may be used to further study
RNA-Immunoprecipitation (RIP) of Pol V transcripts
Using a similar strategy as ChIP, protein–RNA interactions can be detected. When applied to studying interactions between proteins and Pol V-produced non-coding transcripts they provide a link between a protein’s effect on chromatin and the ability to bind lncRNA. As for ChIP, binding is manifested as enrichment between conditions/genotypes when analyzed by real-time RT-PCR. Protein–RNA interactions are covalently fixed using reversible chemical crosslinking. RIP follows the same steps as ChIP:
Real-time RT-PCR of low abundance lncRNA
Following RIP, RNA is converted to cDNA and amplified by real-time PCR. In addition to analyzing RIP, this method may be used for detection of lncRNAs in total RNA samples. Long non-coding RNAs, including Pol V transcripts, are generally transcribed at low levels (some approximately 10,000 fold lower than ACTIN2), causing them to be difficult to detect. This method is sensitive enough to reliably and quantitatively detect Pol V transcripts and other low abundance RNAs. The protocol involves a
ChIP-sequence analysis
ChIP-seq is performed to identify protein binding sites throughout the genome. In order to generate a list of binding locations, the raw sequencing reads must first be processed in a way that accurately maps to the Arabidopsis genome while controlling for quality. After reads have been mapped, enrichment scores can be generated and utilized to obtain genomic coordinates of binding sites (Fig. 4). Several different algorithms exist for each step and decisions on the use of each algorithm can be
Conclusions
The activity of lncRNA can be studied through a combination of techniques focused on indentifying loci controlled by RdDM. New targets of transcriptional gene silencing can be detected by ChIP-seq for proteins that bind chromatin in a way dependent on lncRNA. These loci can then be directly tested for lncRNA production and protein–RNA interactions. Additionally, by using a combination of these methods in various mutant backgrounds and with antibodies for different proteins, molecular mechanisms
Acknowledgements
We thank Brian Gregory and Qi Zheng for sharing expertise in high throughput sequencing data analysis. This work has been supported by National Science Foundation grant MCB 1120271 to A.T.W. and Austrian Science Fund (FWF) fellowship J3199-B09 to G.B.
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