Abstract
BackgroundLong non-coding RNAs (lncRNAs) have been implicated in diverse biological processes. In contrast to extensive genomic annotation of lncRNA transcripts, far fewer have been characterized for subcellular localization and cell-to-cell variability. Addressing this requires systematic, direct visualization of lncRNAs in single cells at single-molecule resolution.ResultsWe use single-molecule RNA-FISH to systematically quantify and categorize the subcellular localization patterns of a representative set of 61 lncRNAs in three different cell types. Our survey yields high-resolution quantification and stringent validation of the number and spatial positions of these lncRNA, with an mRNA set for comparison. Using this highly quantitative image-based dataset, we observe a variety of subcellular localization patterns, ranging from bright sub-nuclear foci to almost exclusively cytoplasmic localization. We also find that the low abundance of lncRNAs observed from cell population measurements cannot be explained by high expression in a small subset of ‘jackpot’ cells. Additionally, nuclear lncRNA foci dissolve during mitosis and become widely dispersed, suggesting these lncRNAs are not mitotic bookmarking factors. Moreover, we see that divergently transcribed lncRNAs do not always correlate with their cognate mRNA, nor do they have a characteristic localization pattern.ConclusionsOur systematic, high-resolution survey of lncRNA localization reveals aspects of lncRNAs that are similar to mRNAs, such as cell-to-cell variability, but also several distinct properties. These characteristics may correspond to particular functional roles. Our study also provides a quantitative description of lncRNAs at the single-cell level and a universally applicable framework for future study and validation of lncRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0586-4) contains supplementary material, which is available to authorized users.
Highlights
Long non-coding RNAs have been implicated in diverse biological processes
A single molecule, single cell RNA RNA fluorescence in situ hybridization (FISH) survey of Long non-coding RNAs (lncRNAs) in three human cell types To characterize the abundance and localization patterns of lncRNAs in the three different cell types, we studied 61 lncRNAs systematically selected to span a range of parameters (Figure 1a) using single molecule RNA FISH
We manually curated a candidate set of 61 lncRNA for screening (Figure 1; Additional files 1 and 2) such that: (1) the lncRNAs in our set are significantly expressed in at least one of human foreskin fibroblasts, human lung a RNA FISH in 3 cell types
Summary
Long non-coding RNAs (lncRNAs) have been implicated in diverse biological processes. In contrast to extensive genomic annotation of lncRNA transcripts, far fewer have been characterized for subcellular localization and cell-to-cell variability. Addressing this requires systematic, direct visualization of lncRNAs in single cells at single-molecule resolution. Deep-sequencing based studies have revealed thousands of long non-coding RNAs (lncRNAs) expressed from mammalian genomes. While a number of studies have implicated functional roles lncRNAs [1,2,3] the vast majority remain uncharacterized [4,5].
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