Research resource: RNA-Seq reveals unique features of the pancreatic β-cell transcriptome - PubMed (original) (raw)

Research resource: RNA-Seq reveals unique features of the pancreatic β-cell transcriptome

Gregory M Ku et al. Mol Endocrinol. 2012 Oct.

Abstract

The pancreatic β-cell is critical for the maintenance of glycemic control. Knowing the compendium of genes expressed in β-cells will further our understanding of this critical cell type and may allow the identification of future antidiabetes drug targets. Here, we report the use of next-generation sequencing to obtain nearly 1 billion reads from the polyadenylated RNA of islets and purified β-cells from mice. These data reveal novel examples of β-cell-specific splicing events, promoter usage, and over 1000 long intergenic noncoding RNA expressed in mouse β-cells. Many of these long intergenic noncoding RNA are β-cell specific, and we hypothesize that this large set of novel RNA may play important roles in β-cell function. Our data demonstrate unique features of the β-cell transcriptome.

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Figures

Fig. 1.

Fig. 1.

mRNA-seq of mouse islets and primary mouse β-cells demonstrates β-cell-specific genes. A, A comparison of RT-qPCR quantification (y-axis) and mRNA-seq quantification (x-axis) of GPCR expression. Each point represents a single GPCR gene. RT-qPCR data were plotted with permission (16). B, Log base 2-fold change in normalized read counts from β-cells vs. islets for the indicated genes. All were statistically significant with q value < 1 × 10−14. C, Identification of RefSeq genes enriched and depleted in β-cells. FPKM in β-cells (y-axis) is plotted _vs._ the FPKM in islets (x-axis). _Red dots_ indicate a q value < 0.1. _Black dots_ indicate q value > 0.1. The blue dashed oval is highly enriched for exocrine secreted enzymes (see text). D, Histogram of FPKM levels of RefSeq genes expressed in β-cells. E, Log base 10 ratio of β-cell FPKM to the average FPKM of all non-β tissues is plotted (β-cell specificity score) for β-cell-expressed genes. Green circles indicate genes statistically significantly increased in β-cells over all five non-β-cell tissues (q value < 0.1). F, FPKM values of the 16 genes with no detectable expression in any of the other five non-β-cell tissues examined. AU, Arbitrary units.

Fig. 2.

Fig. 2.

mRNA-seq identifies the β-cell Tph1 promoter. A, The short form of Tph1 is the dominant isoform expressed in β-cells. mRNA-seq reads from β-cells (top) are plotted with the exon structure of RefSeq transcripts for Tph1 locus (bottom). On the left is a heat map of expression level of each transcript in β-cells and skeletal muscle. B, RT-PCR with primers to the indicated exons was performed and run on an agarose gel. C, RT-qPCR targeting the indicated exons from islets isolated from pregnant (preg) and nonpregnant (non-preg) mice. Fold induction over nonpregnant levels is plotted with

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(n = 2 mice from each condition). *, P value < 0.01 between pregnant and nonpregnant mice. D, The upstream region of exon 1b is sufficient to drive transcription in β-cells. The indicated constructs were transfected into MIN6 cells, and luciferase activity was determined. The

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is plotted (n = 4). *, P < 0.0001 vs. no-promoter control.

Fig. 3.

Fig. 3.

Examples of β-cell-specific splicing events and alternative promoter use. A, Vps8: heat map showing expression of three major isoforms of Vps8 (left) and their transcript structures (bottom right). mRNA-seq reads mapping to the locus are shown for β-cell, liver, lung fibroblast (fibro), and neural progenitor cell (NPC) (top). The green box highlights a β-cell-specific exon 1 that is not present in other tissues. B, Rasgrf1: as in A, but Rasgrf1 is expressed only in brain and β-cells, so only these two tissues are listed. The green box highlights a β-cell-specific exon 2.

Fig. 4.

Fig. 4.

Identification of β-cell lincRNA. A, Filtering strategy for lincRNA identification (see text for details). B, Cumulative distribution plot of FPKM of lincRNA (red) and protein-coding (green) genes shows that most lincRNA are expressed at lower levels vs. protein-coding genes. C, β-Cell enrichment score (log base 10 ratio of β-cell FPKM over average non-β-cell FPKM) for lincRNA (black and red) and protein-coding genes (black and green). Colored circles indicate genes for which there is a statistically significant increase in β-cells over all six non-β-cell tissues examined. D, Histogram of maximum PhyloP score (placental mammals) of lincRNA (red), protein-coding genes (NM, blue), and repeat masked sequence (RPMSK, black) showing that most protein-coding genes show high conservation, whereas only a minority of lincRNA show equivalent conservation. E, mRNA-seq reads and predicted transcript structure of a novel potential lincRNA 5′ of the Nkx6-1 locus. F, mRNA-seq reads and predicted transcript structure of lying antisense to the Pdx1 locus. NPC, Neural progenitor cell; sk muscl, skeletal muscle.

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