Identification of Non-Coding RNAs Associated with Telomeres Using a Combination of enChIP and RNA Sequencing

Abstract

Accumulating evidence suggests that RNAs interacting with genomic regions play important roles in the regulation of genome functions, including X chromosome inactivation and gene expression. However, to our knowledge, no non-biased methods of identifying RNAs that interact with a specific genomic region have been reported. Here, we used enChIP-RNA-Seq, a combination of engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) and RNA sequencing (RNA-Seq), to perform a non-biased search for RNAs interacting with telomeres. In enChIP-RNA-Seq, the target genomic regions are captured using an engineered DNA-binding molecule such as a transcription activator-like protein. Subsequently, RNAs that interact with the target genomic regions are purified and sequenced. The RNAs detected by enChIP-RNA-Seq contained known telomere-binding RNAs, including the telomerase RNA component (Terc), the RNA component of mitochondrial RNA processing endoribonuclease (Rmrp), and Cajal body-specific RNAs. In addition, a number of novel telomere-binding non-coding RNAs were also identified. Binding of two candidate non-coding RNAs to telomeres was confirmed by immunofluorescence microscopy and RNA fluorescence in situ hybridization (RNA-FISH) analyses. The novel telomere-binding non-coding RNAs identified here may play important roles in telomere functions. To our knowledge, this study is the first non-biased identification of RNAs associated with specific genomic regions. The results presented here suggest that enChIP-RNA-Seq analyses are useful for the identification of RNAs interacting with specific genomic regions, and may help to contribute to current understanding of the regulation of genome functions.

Identification of physical interactions between genomic regions by enChIP‐Seq

“…) (see review (Fujita & Fujii )) and engineered DNA‐binding molecule‐mediated ChIP (enChIP) (Fujita & Fujii , ; Fujita et al . , , ,b) (see reviews (Fujii & Fujita ; Fujita & Fujii )). enChIP consists of the following steps (Fig.…”

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

“…Using enChIP analyses, we were able to successfully identify a number of known and novel molecules interacting with specific genomic regions (Tables 1-3) [14][15][16][17] . However, this technique failed to detect some other known proteins interacting with these regions.…”

“…Express 3×FLAG-dCas9 and the gRNA (CRISPR-based procedure) or 3×FLAG-TAL (TAL protein-based procedure) in the cells to be analyzed as described previously [14][15][16][17] . 1.…”

“…Confirm the expression of the 3xFLAG-dCas9 or 3xFLAG-TAL protein in the transfected cells by immunoblot analysis using an anti-FLAG antibody (Ab) as described previously [14][15][16][17] . Note: Expression of these tagged proteins can also be confirmed by intracellular staining with anti-FLAG-FITC and a subsequent FACS analysis.…”

“…In order to perform biochemical analysis of specific genomic regions easily, we have developed two locus-specific chromatin immunoprecipitation (ChIP) technologies, namely insertional ChIP (iChIP) [8][9][10][11][12][13] and engineered DNA-binding molecule-mediated ChIP (enChIP) [14][15][16][17] . In iChIP, a locus of interest is tagged by inserting recognition sequences of an exogenous DNA-binding protein such as LexA.…”