Abstract
An increasing number of long noncoding RNAs (lncRNAs) have experimentally confirmed functions, yet little is known about their transcriptional dynamics and it is challenging to determine their regulatory effects. Here, we used allele-sensitive single-cell RNA sequencing to demonstrate that, compared to messenger RNAs, lncRNAs have twice as long duration between two transcriptional bursts. Additionally, we observed increased cell-to-cell variability in lncRNA expression due to lower frequency bursting producing larger numbers of RNA molecules. Exploiting heterogeneity in asynchronously growing cells, we identified and experimentally validated lncRNAs with cell state-specific functions involved in cell cycle progression and apoptosis. Finally, we identified cis-functioning lncRNAs and showed that knockdown of these lncRNAs modulated the nearby protein-coding gene’s transcriptional burst frequency or size. In summary, we identified distinct transcriptional regulation of lncRNAs and demonstrated a role for lncRNAs in the regulation of mRNA transcriptional bursting.
Highlights
An increasing number of long noncoding RNAs have experimentally confirmed functions, yet little is known about their transcriptional dynamics and it is challenging to determine their regulatory effects
The detection of hundreds of long noncoding RNAs (lncRNAs) per cell motivated us to proceed with in-depth investigations of lncRNA expression across cells
We initially excluded lncRNAs and mRNAs that had another promoter within 4 kilobases since we noticed that genes with closely located promoters had increased expression (Extended Data Fig. 1d,e, referred to as separated transcriptional units)
Summary
An increasing number of long noncoding RNAs (lncRNAs) have experimentally confirmed functions, yet little is known about their transcriptional dynamics and it is challenging to determine their regulatory effects. We observed increased cell-to-cell variability in lncRNA expression due to lower frequency bursting producing larger numbers of RNA molecules. Analyses of transcriptional bursting to date have focused on protein-coding genes and it is unknown whether the low expression of lncRNAs is mediated by lowered burst sizes (fewer RNA molecules per cell) or burst frequencies (expression in fewer cells). Comprehensive analyses of transcriptional dynamics and cell-to-cell variability of lncRNAs are still missing and most studies to date were limited to low throughput methods measuring limited numbers of genes and cells. The introduction of single-cell RNA sequencing (scRNA-seq) technologies and protocols for allele-specific quantification offers new opportunities to characterize transcriptional dynamics and allele-specific gene expression in individual cells for thousands of genes simultaneously. We introduce allele-sensitive scRNA-seq of lncRNAs to investigate lncRNA transcriptional bursting kinetics and identify lncRNA candidates with roles in cellular processes and transcriptional regulation
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