Characteristics of long non-coding RNAs in the Brown Norway rat and alterations in the Dahl salt-sensitive rat

Abstract

Long non-coding RNAs (lncRNAs) are potentially important mediators of genomic regulation. lncRNAs, however, remain poorly characterized in the rat model organism widely used in biomedical research. Using poly(A)-independent and strand-specific RNA-seq, we identified 1,500 to 1,800 lncRNAs expressed in each of the following tissues of Brown Norway rats: the renal cortex, renal outer medulla, liver, cardiac left ventricle, adrenal gland, and hypothalamus. Expression and the binding of histone H3K4me3 to promoter regions were confirmed for several lncRNAs. Rat lncRNA expression appeared to be more tissue-specific than mRNA. Rat lncRNAs had 4.5 times fewer exons and 29% shorter transcripts than mRNA. The median cumulative abundance of rat lncRNAs was 53% of that of mRNA. Approximately 28% of the lncRNAs identified in the renal outer medulla appeared to lack a poly(A) tail. Differential expression of 74 lncRNAs was detected in the renal outer medulla between Dahl SS rats, a model of salt-sensitive hypertension, and salt-insensitive, congenic SS.13(BN26) rats fed a high-salt diet. Two of the differentially expressed lncRNAs, which were confirmed, were located within the congenic region and contained several sequence variants. The study identified genome-wide characteristics of lncRNAs in the rat model and suggested a role of lncRNAs in hypertension.

Figure 1. Classification of the identified rat lncRNAs.

A. Classification of lncRNAs identified in at least one of the six tissues obtained from Brown Norway rats. lncRNAs were classified as antisense, intronic complete overlap (concs), intronic contained (incs), intronic partial overlap (poncs), and intergenic based on the spatial relationship between the genomic sequences encoding lncRNAs and protein-coding genes. B. Schematic illustration of the classification of lncRNA genes based on their spatial relationship with protein-coding genes. Black blocks and lines represent exons and introns, respectively, of protein-coding genes. Blocks and lines shown in color represent exons and introns, respectively, of lncRNA genes. Arrows indicate directions of transcription.

Figure 2. Real-time PCR verification of rat lncRNA expression identified by RNA-seq.

Transcript abundance based on RNA-seq (left) and real-time PCR (right) is shown for an lncRNA identified by RNA-seq as enriched in each of the tissues. Li, liver; LV, cardiac left ventricle; C, renal cortex; M, renal outer medulla; H, hypothalamus; A, adrenal gland. n = 4 for real-time PCR. *, P<0.05 vs. the tissue in which the lncRNA was enriched based on RNA-seq.

Figure 3. Higher transcript abundance for rat lncRNA genes was often associated with increased H3K4me3 binding to promoter regions.

A. Chromatin fragments immunoprecipitated from five rat tissues using an antibody for histone H3K4me3 were enriched for a GAPDH promoter region, but not a myoglobin exon region. Promoter regions for lncRNAs with higher transcript abundance in the liver (B), cardiac left ventricle (C), renal cortex (D), or renal outer medulla (E) were enriched in chromatin fragments immunoprecipitated from the respective tissue using an antibody for histone H3K4me3. The promoter enrichment was minimal for an lncRNA with higher transcript abundance in the hypothalamus (F). The lncRNAs for which promoter data are shown in the current figure correspond to those for which transcript abundance data are shown in Figure 2. Li, liver; LV, cardiac left ventricle; C, renal cortex; M, renal outer medulla; H, hypothalamus; A, adrenal gland.

Figure 4. Expression of rat lncRNAs was tissue-specific.

A. A heat map of lncRNA expression in the six tissues. Each sample (i.e., each row in the heat map) was a pool of two rats. B. Proportions of lncRNA or mRNA transcripts found in one or more of the six tissues examined. Li, liver; LV, cardiac left ventricle; C, renal cortex; M, renal outer medulla; H, hypothalamus; A, adrenal gland.

Figure 5. Exon number, transcript length, and abundance of rat lncRNA compared to mRNA.

Rat lncRNA transcripts had fewer exons (A), modestly shorter lengths (B), and lower abundance (C) compared to mRNA.

Figure 6. lncRNAs differentially expressed between SS and congenic SS.13^BN26 rats and located on the congenic region.

Abundance levels based on RNA-seq (left) and real-time PCR (right) are shown for two lncRNAs (A, B) located in the congenic region of SS.13^BN26. LS, 0.4% NaCl diet; HS, 4% NaCl diet for 7 days; L26, SS.13^BN26 rat. n = 6–7 for real-time PCR. *, P<0.05 vs. SSLS; #, P<0.05 vs. SSHS. C. Genomic locations of the two lncRNAs. Nucleotides 75,226,423–77,023,203 of chromosome 13, indicated by the red square, are shown in detail. The two lncRNAs are indicated by red ovals.