Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [RIP-Seq]

To identify potential lncRNAs that associate with CENP-C, RNA immunoprecipitation (RIP) using the CENP-C and control IgG antibody was performed in the whole-cell lysates of HeLa cells, followed by Illumina short-read sequencing (RIP-seq). We found three CENP-C-binding lncRNAs that meet the enrichment criteria (fold change > 2, p < 0.01), including AGAP2-AS1, GS1-124K5.4, and AC124789.1 (lnc-CCTT) . Consistent with the results of RIP-seq, RIP-qPCR assay in whole-cell lysates showed that all three lncRNAs could bind to CENP-C, while lnc-CCTT showing the most robust association of CENP-C. ). It is well-established that the localization of lncRNAs within the cell is the primary determinant of their molecular functions. RT-qPCR (quantitative PCR with reverse transcription) assay of subcellular fractionation and RNA fluorescence in situ hybridization (RNA-FISH) revealed that AGAP2-AS1 and GS1-124K5.4 dominantly distributed in the cytoplasm, whereas lnc-CCTT, specifically and almost exclusively, localized to the nucleus. Thus, lnc-CCTT appears to be a prime interacting RNA of CENP-C at centromere. Identification and characterization of non-coding RNAs associated with CENPC in Hela cells