Single-cell profiling of lncRNAs and their cell lineage commitment roles via RNA-DNA-DNA triplex formation in mammary epithelium.

Long noncoding RNAs (lncRNAs), which are crucial for organ development, exhibit cell-specific expression. Thus, transcriptomic analysis based on total tissue (bulk-seq) cannot accurately reflect the expression pattern of lncRNAs. Here, we used high-throughput single-cell RNA-seq data to investigate the role of lncRNAs using the hierarchical model of mammary epithelium. With our comprehensive annotation of the mammary epithelium, lncRNAs showed much greater cell-lineage specific expression than coding genes. The lineage-specific lncRNAs were functionally correlated with lineage commitment through the coding genes via thecis- andtrans-effects of lncRNAs. For the working mechanism, lncRNAs formed a triplex structure with the DNA helix to regulate downstream lineage-specific marker genes. We used lncRNA-Carmnas an example to validate the above findings.Carmn, which is specifically expressed in mammary gland stem cells (MaSCs) and basal cells, positively regulated the Wnt signaling ligandWnt10athrough formation of a lncRNA-DNA-DNA triplex, and thus controlled the stemness of MaSCs. Our study suggests that lncRNAs play essential roles in cell-lineage commitment and provides an approach to decipher lncRNA functions based on single-cell RNA-seq data.