Global analysis of biogenesis, stability and sub-cellular localization of lncRNAs mapping to intragenic regions of the human genome

Abstract

Long noncoding RNAs (lncRNAs) that map to intragenic regions of the human genome with the same (intronic lncRNAs) or opposite orientation (antisense lncRNAs) relative to protein-coding mRNAs have been largely dismissed from biochemical and functional characterization due to the belief that they are mRNA precursors, byproducts of RNA splicing or simply transcriptional noise. In this work, we used a custom microarray to investigate aspects of the biogenesis, processing, stability, evolutionary conservation, and cellular localization of ∼ 6,000 intronic lncRNAs and ∼ 10,000 antisense lncRNAs. Most intronic (2,903 of 3,427, 85%) and antisense lncRNAs (4,945 of 5,214, 95%) expressed in HeLa cells showed evidence of 5' cap modification, compatible with their transcription by RNAP II. Antisense lncRNAs (median t1/2 = 3.9 h) were significantly (p < 0.0001) more stable than mRNAs (median t1/2 = 3.2 h), whereas intronic lncRNAs (median t1/2 = 2.1 h) comprised a more heterogeneous class that included both stable (t1/2 > 3 h) and unstable (t1/2 < 1 h) transcripts. Intragenic lncRNAs display evidence of evolutionary conservation, have little/no coding potential and were ubiquitously detected in the cytoplasm. Notably, a fraction of the intronic and antisense lncRNAs (13 and 15%, respectively) were expressed from loci at which the corresponding host mRNA was not detected. The abundances of a subset of intronic/antisense lncRNAs were correlated (r ≥ |0.8|) with those of genes encoding proteins involved in cell division and DNA replication. Taken together, the findings of this study contribute novel biochemical and genomic information regarding intronic and antisense lncRNAs, supporting the notion that these classes include independently transcribed RNAs with potentials for exerting regulatory functions in the cell.

Keywords: Intronic lncRNAs; RNA stability; RNA subcellular localization; antisense lncRNAs; eukaryotic transcription.

Figure 1.

Stability and sub-cellular localization of lncRNAs and mRNAs. (A) Schematic representation of the intronic (red) and antisense (blue) lncRNAs and protein-coding mRNAs (green) present in intron-exon oligoarray. (B) The distribution of half-lives of the lncRNAs and mRNAs following transcription inhibition with actinomycin D at different time points (1, 3, 6, and 8 h). (C) Aliquots of total RNA were digested with 5′-phosphate-dependent exonuclease (5′-exo) with or without tobacco acid pyrophosphatase (TAP) pre-treatment to release the 5′ cap and render the RNA susceptible to 5′ exonuclease digestion. The numbers of intronic lncRNAs, antisense lncRNAs and mRNAs with or without a 5′ cap (y-axis) were determined by identifying those that were significantly affected by digestion with both enzymes (TAP⁺/5′-exo⁺) compared with controls digested with 5′-exo only (TAP⁻/5′-exo⁺), and these numbers were plotted according to RNA stability (x-axis). The data reflect results obtained from 4 independent replicates of 5′ cap assay. (***) Fisher's test, p < 0.0002. (D) Relative distributions of lncRNAs and mRNAs in nuclear (Nc) and cytoplasmic (Cyt) fractions of HeLa cells, as determined by 2 independent replicate measurements.

Figure 2.

Cell type specificity of lncRNAs and mRNAs. Venn diagrams showing the expression overlap of (A) intronic lncRNAs, (B) antisense lncRNAs, and (C) mRNAs detected in each of the 4 cell lineages Mia PaCa 2, DU-145, MCF-7, and HeLa. (D) The fractional expression levels (FELs) of the lncRNAs (intronic, n = 3,937; antisense, n = 5,889) and mRNAs (n = 5,866) across the 4 cell lineages were calculated (see Methods for details). The percentages of transcripts with an FEL higher or lower than 0.5 in each class are shown. Higher FEL values correspond with a more highly cell-specific expression pattern. The observed fractions of cell-specific intronic and antisense lncRNAs are significantly different compared with those of the mRNAs. (***) Chi-square test, p < 0.0001. The data reflect results obtained from 2 independent replicates.

Figure 3.

Association of intronic and antisense lncRNA loci with regulatory chromatin marks. Distance distributions of (A) H3K4me3, (B) H3K4me1, (C) H3K27Ac, and (D) CpG islands within a 10 kb window relative to the genomic coordinates of the predicted TSSs of intronic (red line) and antisense (blue line) lncRNAs and mRNAs (green line) expressed in at least one of the 4 cell lineages studied. Ten random sets of sequences with the same numbers, lengths and genomic contexts were used as negative controls (gray lines, see Methods for details). The mRNA and intronic and antisense lncRNA distributions were significantly different relative to the average distributions for the random control sequences (Kolmogorov-Smirnov test, p < 0.005).

Figure 4.

Conservation analysis of intronic and antisense lncRNAs. (A and E) TransMap cross-species cDNA alignments of 15 vertebrate species with the human genome (species indicated in the rows) overlapping (A) 512 (13% of total) intronic lncRNAs and (E) 1,988 (34% of total) antisense lncRNAs expressed in at least one cell line investigated in this study and with conserved expression in at least one species (red and blue dashes show expression conservation). Higher proportions of expression conservation of the intronic (A) and antisense (B) lncRNA datasets were found compared with 10 control data sets of randomly selected sequences with the same numbers, transcript lengths and genomic context (Chi-square test, p < 0.01). (B and F) Number of human intronic (B) or antisense (F) lncRNAs with sequence similarity to ESTs from selected vertebrate, mammalian and primate species. The same number of randomly selected ESTs from each species was used to avoid sampling bias. (C and G) DNA sequence conservation of intragenic lncRNAs within vertebrate, mammal and primate groups. The asterisks show statistically significant differences in the numbers of conserved PhastCons elements overlapping intronic (C) and antisense (G) lncRNAs compared with 10 sets of randomly selected genomic regions with same numbers, lengths and genomic context (gray bars, *** Chi-square test, p <0.0001). (D and H) Venn diagram with overlaps determined by 3 different conservation analyses of intronic (D) and antisense (H) lncRNAs. RNAz predicted secondary structure conservation for 97 and 164, PhastCons predicted DNA conservation for 2,036 and 4,545, and TransMap predicted expression conservation for 512 and 1,988 intronic and antisense lncRNAs, respectively.

Figure 5.

Co-expression between lncRNAs and mRNAs from the same locus. Distribution of Pearson correlation coefficients between the intronic (A) or antisense lncRNAs (B) and mRNAs expressed from the same gene locus across 4 cell lineages (black bars). As a control, correlations were also calculated following the shuffling of lncRNA sequences across gene loci (gray bars). Significant differences in the correlation distributions of lncRNA/mRNA expressed from the same locus and from shuffled loci were determined using the Kolmogorov-Smirnov nonparametric test (p < 0.0001).