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. 2014 Jan 7;15(1):R6.
doi: 10.1186/gb-2014-15-1-r6.

Extensive Localization of Long Noncoding RNAs to the Cytosol and Mono- And Polyribosomal Complexes

Free PMC article

Extensive Localization of Long Noncoding RNAs to the Cytosol and Mono- And Polyribosomal Complexes

Sebastiaan van Heesch et al. Genome Biol. .
Free PMC article

Abstract

Background: Long noncoding RNAs (lncRNAs) form an abundant class of transcripts, but the function of the majority of them remains elusive. While it has been shown that some lncRNAs are bound by ribosomes, it has also been convincingly demonstrated that these transcripts do not code for proteins. To obtain a comprehensive understanding of the extent to which lncRNAs bind ribosomes, we performed systematic RNA sequencing on ribosome-associated RNA pools obtained through ribosomal fractionation and compared the RNA content with nuclear and (non-ribosome bound) cytosolic RNA pools.

Results: The RNA composition of the subcellular fractions differs significantly from each other, but lncRNAs are found in all locations. A subset of specific lncRNAs is enriched in the nucleus but surprisingly the majority is enriched in the cytosol and in ribosomal fractions. The ribosomal enriched lncRNAs include H19 and TUG1.

Conclusions: Most studies on lncRNAs have focused on the regulatory function of these transcripts in the nucleus. We demonstrate that only a minority of all lncRNAs are nuclear enriched. Our findings suggest that many lncRNAs may have a function in cytoplasmic processes, and in particular in ribosome complexes.

Figures

Figure 1
Figure 1
Experimental workflow and quality control. (A) Cells were lysed and the complete cytosolic fraction was used for ribosomal fractionation. Pelleted nuclei and nine fractions (indicated A to I) derived from the ribosomal fractionation were subsequently used for RNA isolation and strand-specific RNA-seq. Fractions A1 and A2 as well as B1 and B2 were merged prior to the RNA-seq. (B) 2100 Bioanalyzer RNA 6000 Pico results showing the integrity of the collected RNA samples obtained by ribosomal fractionation. Each ribosomal fraction has an RNA integrity value of 10. These results also show the sample-specific content of tRNAs, 5S, 5.8S, 18S and 28S rRNA, which nicely indicate the purity of the fractionation. RIN, RNA integrity.
Figure 2
Figure 2
Subcellular RNA fractions have a different transcript composition. (A) Scatter plot and correlation matrix of all sequenced samples. The color intensity of the correlation boxes (r values) depicts the relative strength of the correlation, ranging between 0.39 and 0.97. (B) RNA species content of each sequenced fraction in counts per million. CPM, counts per million; lincRNA, long intergenic noncoding RNA; snoRNA, small nucleolar RNA; snRNA, small nuclear RNA.
Figure 3
Figure 3
Long noncoding RNAs show a subcellular distribution similar to specific groups of protein-coding transcripts. Heatmap of the Spearman-Rank correlation between the each of the 152 expressed lncRNAs and 7,206 expressed protein-coding transcripts across the subcellular RNA samples. Strong correlations are shown in blue, anti-correlations are shown in red. Six frequently studied lncRNAs with varying correlations to protein-coding transcripts are highlighted at the bottom together with a large cluster that harbors the majority of expressed snoRNA host genes. lncRNA, long noncoding RNA.
Figure 4
Figure 4
RNA species show specific distributions across the subcellular RNA samples. (A) Heatmap display of the 11 clusters and the number of protein-coding, lncRNA and sncRNA transcripts present in each cluster. (B) Summarizing plot showing the distribution of the three types of transcripts over the four major types of clusters that could be derived from the analysis in (A). (C) Boxplots of the total transcript length and the maximum (potential) open reading frame of protein-coding transcripts and lncRNAs in clusters VI to X. lncRNA, long noncoding RNA; ORF, open reading frame; sncRNA, short noncoding RNA.
Figure 5
Figure 5
Individual long noncoding RNAs are differentially distributed across subcellular samples. The normalized read counts of seven lncRNAs that are found in different clusters in Figure 4. CPM, counts per million.

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