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

Long non-coding 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 the cis- and trans-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-Carmn as 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 ligand Wnt10a through 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. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: By coupling multiple database originated gene annotation files and the bulk-seq assembled transcriptome, researchers could use the high throughput scRNA-seq technology, for example, Drop-seq, 10X Genomic scRNA-seq, to understand the temporal- and cell type- specific of lncRNAs, concurrently include numerous previously unannotated lncRNAs, in the mammary gland epithelium. The expression specificity of lncRNAs can be used to demarcate sub-populations of mammary epithelium. By systematically integrated analysis the expression correlation of lncRNA-mRNA and the lncRNA-DNA-DNA triplex formation potency, it shows lineage-specific lncRNAs can regulate lineage commitment through formation of a lncRNA-DNA-DNA triplex, pervasively, and thus control the stemness of mammary stem cells (MaSCs). This study provides an approach to decipher lncRNA functions by incorporating scRNA-seq with bulk-seq data.