Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution - PubMed (original) (raw)

Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution

Moran N Cabili et al. Genome Biol. 2015.

Abstract

Background: 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.

Results: We 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.

Conclusions: Our 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.

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Figures

Figure 1

Figure 1

An RNA-FISH survey of lncRNAs. (a) Study workflow. (b) Key features of 61 lincRNAs for which probe sets were successfully designed and were imaged in the study. Shown are for each of 61 lincRNA (column) the following features from top to bottom: whether it has a syntenic ortholog (black: has ortholog) or a divergently transcribed mRNA neighbor (black: has such neighbor), the extent of tissue specificity across 23 tissues (blue color intensity: maximal tissue specificity score as in [9] across the tissues presented in the figure; white to blue color bar), its expression level as measured by RNA-Seq (red intensity: the fractional density across the row of log2(FPKM) as estimated by Cufflinks; white to red color bar) in each of 23 tissues (heatmap rows; Additional file 2), and the extent of analysis performed (black: lncRNAs with valid probe set that were included in the final analysis; white: lncRNAs showing no signal; gray: lncRNAs with an invalid probe based on the two-color co-localization assay).

Figure 2

Figure 2

LncRNAs exhibit a variety of cellular localization patterns. Florescence micrographs of representative expressing cells for each of 34 lncRNAs with a validated probe set. LncRNA-cell pairs are classified to cellular localization types I to V as described in the Methods (marked by their border color). Magenta stars mark five lncRNAs that are presented in two different cell types and two different classes (see same row for comparison). Scale bar, 5 μm; when a scale bar is not specified, reference the scale bar within the top left image. Top panel: fraction of each classification for each type across the full set of 70 valid lncRNA-cell pairs imaged.

Figure 3

Figure 3

Most lincRNAs are predominantly localized to the nucleus. (a) Boxplots describing the distribution of the fraction of molecules localized to the nucleus (Y axis) for each validated lncRNA-cell pair (X axis, orange: HeLa, blue: hFF, purple: hLF). Red bar: medians. Whiskers are at 1.5* the inner quartile range. (b) Scatter plot of the relationship between expression level (X axis; median number of molecules per cell) and nuclear localization (Y axis, median fraction of nuclear spots across all expressing cells). Each data point is one gene-cell pair and is colored by its classification to the localization classes I to V (Methods) of Figure 2. mRNA sets 1 to 2 (yellow) serve as a reference. Histograms on top and right are the distribution of all lncRNAs- (black) and mRNA- (yellow) cell pairs. (c) Scatter and histograms as in (b) but for lncRNA with (red) or without (black) a divergently transcribed mRNA counterpart. (d) Scatter and histograms as (b) but for lncRNA with (red) or without (black) a syntenic ortholog. (e) Representative image of mitotic cells (marked with white arrows) lacking foci that are seen in interphase cells (marked with yellow arrows). Scale bar, 5 μm.

Figure 4

Figure 4

Cell-to-cell variability does not appear to explain the low abundance of the lncRNAs in our survey. (a) Distribution of RNA single molecules counts (bins, columns; Red intensity: fractional density of molecule counts across the population) for the 64 lncRNA-cell pairs in the validated set that are quantitative (rows, Methods). Cell type color coding: orange - HeLa, blue - hFF, purple - hLF. Left bins are sized 3 (0 to 50 molecules ), where right in bins are sized 10 (50 to 300 molecules). A heterogeneously expressed lncRNA (XLOC_003526) and a homogenously expressed lncRNA (XLOC_006922), are pointed by black arrows and referenced in figures b and c. (b, c) The relationship between the mean molecule count (X axis) vs. median molecule count (Y axis, b) or vs. variability in molecule counts (Y axis, coefficient of variation, c) for the 64 lncRNA-cell pairs in the quantitative validated set (red), mRNA set 1 (green circles; Methods) and mRNA set 2 (green diamonds; Methods). A linear regression line in b (black) supports the consistency of the majority of transcript-cell pairs with a unimodal distribution (Y = 0.87X-1.25, Pearson r = 0.96). Dotted line is Y = X. Black curve in (c) is the theoretic Poisson distribution. Four transcripts marked (1 to 4) are analyzed further in d and e. LncRNA pairs with mean >170 (less than 10% of all pairs) are not presented, but show a similar pattern on a log scale. (d) Fluorescence micrographs of single molecule RNA FISH of a homogenously expressed lncRNA (1-XLOC_006922; top left) and mRNA (2-FOXF1; top right) and of a heterogeneously expressed lncRNA (3- XLOC_003526; bottom left) and mRNA (4 - CCNA2; bottom right). XLOC_003526 and CCNA2 are both heterogeneous but do not correlate with each other based on co-staining in two colors. Scale bar, 5 μm. (e) Molecule count distributions for each of the example transcript 1 to 4.

Figure 5

Figure 5

Cellular localization of divergent lincRNAs and their neighbors. (a) Two-color overlay micrograph presenting florescence probes targeting the lincRNA (green) and coding neighbor (red). Co-localized spots are marked yellow. The lincRNA and cell type are marked on the image. Scale bar, 5 μm; marked on the left most image. Top: illustration of the positional genomic orientation of a divergent lincRNA and its coding gene neighbor. (b) Representative fluorescence micrographs as shown in Figure 2 for the lincRNAs in a. Scale bar, 5 μm. (c) Scatter plots of the relationship in each cell between the expression level of the lincRNA (X axis, molecule count) and that of its neighboring coding gene (Y axis). Pearson correlation coefficients (r) after removal of outliers (Additional file 1) are denoted on top. Data in (a-c) are presented for eight of the nine lincRNA-gene neighbor pairs for which a valid probe set exists.

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References

    1. Rinn JL, Chang HY. Genome regulation by long noncoding RNAs. Annu Rev Biochem. 2012;81:145–166. doi: 10.1146/annurev-biochem-051410-092902. - DOI - PMC - PubMed
    1. Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev Genet. 2009;10:155–159. doi: 10.1038/nrg2521. - DOI - PubMed
    1. Sauvageau M, Goff LA, Lodato S, Bonev B, Groff AF, Gerhardinger C, et al. Multiple knockout mouse models reveal lincRNAs are required for life and brain development. eLife. 2013;2:e01749. doi: 10.7554/eLife.01749. - DOI - PMC - PubMed
    1. Ulitsky I, Bartel DP. lincRNAs: genomics, evolution, and mechanisms. Cell. 2013;154:26–46. - PMC - PubMed
    1. Kung JT, Colognori D, Lee JT. Long noncoding RNAs: past, present, and future. Genetics. 2013;193:651–669. doi: 10.1534/genetics.112.146704. - DOI - PMC - PubMed

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