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Transcriptome analysis reveals differential splicing events in IPF lung tissue.

ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a complex disease in which a multitude of proteins and networks are disrupted. Interrogation of the transcriptome through RNA sequencing (RNA-Seq) enables the determination of genes whose differential expression is most significant in IPF, as well as the detection of alternative splicing events which are not easily observed with traditional microarray experiments. We sequenced messenger RNA from 8 IPF lung samples and 7 healthy controls on an Illumina HiSeq 2000, and found evidence for substantial differential gene expression and differential splicing. 873 genes were differentially expressed in IPF (FDR<5%), and 440 unique genes had significant differential splicing events in at least one exonic region (FDR<5%). We used qPCR to validate the differential exon usage in the second and third most significant exonic regions, in the genes COL6A3 (RNA-Seq adjusted pval?=?7.18e-10) and POSTN (RNA-Seq adjusted pval?=?2.06e-09), which encode the extracellular matrix proteins collagen alpha-3(VI) and periostin. The increased gene-level expression of periostin has been associated with IPF and its clinical progression, but its differential splicing has not been studied in the context of this disease. Our results suggest that alternative splicing of these and other genes may be involved in the pathogenesis of IPF. We have developed an interactive web application which allows users to explore the results of our RNA-Seq experiment, as well as those of two previously published microarray experiments, and we hope that this will serve as a resource for future investigations of gene regulation in IPF.

SUBMITTER: Nance T

PROVIDER: S-EPMC3960165 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Transcriptome analysis reveals differential splicing events in IPF lung tissue.

Nance Tracy T Smith Kevin S KS Anaya Vanessa V Richardson Rhea R Ho Lawrence L Pala Mauro M Mostafavi Sara S Battle Alexis A Feghali-Bostwick Carol C Rosen Glenn G Montgomery Stephen B SB

PloS one 20140319 3

Idiopathic pulmonary fibrosis (IPF) is a complex disease in which a multitude of proteins and networks are disrupted. Interrogation of the transcriptome through RNA sequencing (RNA-Seq) enables the determination of genes whose differential expression is most significant in IPF, as well as the detection of alternative splicing events which are not easily observed with traditional microarray experiments. We sequenced messenger RNA from 8 IPF lung samples and 7 healthy controls on an Illumina HiSeq ...[more]

PMID: 24647608

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Project description:Objectives: Idiopathic pulmonary fibrosis (IPF) is a complex disease in which a multitude of proteins and networks are disrupted. Interrogation of genome-wide transcription through RNA sequencing (RNA-Seq) enables the determination of genes whose differential expression is most significant in IPF, as well as the detection of alternative splicing events which are not easily observed with traditional microarray experiments. Methods: Messenger RNA extracted from 8 IPF lung samples and 7 healthy controls was sequenced on an Illumina HiSeq. Analysis of differential expression and exon usage was performed using Bioconductor packages. The gene periostin was selected for validation of alternative splicing by quantitative PCR, and pathway analysis was performed to determine enrichment for differentially expressed and spliced genes. Results: There were 873 genes differentially expressed in IPF (FDR 5%), and 440 unique genes had significant differential splicing events (FDR 5%). In particular, cassette exon 21 of the gene periostin was significantly more likely to be spliced out in IPF samples (adj pval = 2.06e-09), and this result was confirmed by qPCR (Wilcoxon pval = 3.11e-4). We also found that genes close to SNPs in the discovery set of a recent IPF GWAS were enriched for genes differentially expressed in our data, including genes like mucin5B and desmoplakin which have been previously associated with IPF. Conclusions: There is significant differential splicing and expression in IPF lung samples as compared with healthy controls. We found a strong signal of differential cassette exon usage in periostin, an extracellular matrix protein whose increased gene-level expression has been associated with IPF and its clinical progression, but for which differential splicing has not been studied in the context of IPF. Our results suggest that alternative splicing of periostin and other genes may be involved in the pathogenesis of IPF. mRNA sequencing of 8 IPF and 7 control lung tissue samples.

Project description:Alternative splicing is an important way of regulating gene functions in eukaryotes. Several key genes involved in sex determination and gonadal differentiation, such as nr5a1 and ddx4, have sex-biased transcripts between males and females, suggesting a potential regulatory role of alternative splicing in gonads. Currently, the sex-specific alternative splicing events and genes have not been comprehensively studied at the genome-wide level in zebrafish. In this study, through global splicing analysis on three independent sets of RNA-seq data from matched zebrafish testes and ovaries, we identified 120 differentially spliced genes shared by the three datasets, most of which haven't been reported before. Functional enrichment analysis showed that the GO terms of mRNA processing, mRNA metabolism and microtubule-based process were strongly enriched. The testis- and ovary-biased alternative splicing genes were identified, and part of them (tp53bp1, tpx2, mapre1a, kif2c, and ncoa5) were further validated by RT-PCR. Sequence characteristics analysis suggested that the lengths, GC contents, and splice site strengths of the alternative exons or introns may have different influences in different types of alternative splicing events. Interestingly, we identified an unexpected high proportion (over 70%) of non-frameshift exon-skipping events, suggesting that in these cases the two protein isoforms derived from alternative splicing may both have functions. Furthermore, as a representative example, we found that the alternative splicing of ncoa5 causes the loss of a conserved RRM domain in the short transcript predominantly produced in testes. Our study discovers novel sex-specific alternative splicing events and genes with high reliabilities in zebrafish testes and ovaries, which would provide attractive targets for follow-up studies to reveal the biological significances of alternative splicing events and genes in sex determination and gonadal differentiation.

Dataset Information

Transcriptome analysis reveals differential splicing events in IPF lung tissue.

Publications

Transcriptome analysis reveals differential splicing events in IPF lung tissue.

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