RNA sequencing of the exercise transcriptome in equine athletes

Abstract

The horse is an optimal model organism for studying the genomic response to exercise-induced stress, due to its natural aptitude for athletic performance and the relative homogeneity of its genetic and environmental backgrounds. Here, we applied RNA-sequencing analysis through the use of SOLiD technology in an experimental framework centered on exercise-induced stress during endurance races in equine athletes. We monitored the transcriptional landscape by comparing gene expression levels between animals at rest and after competition. Overall, we observed a shift from coding to non-coding regions, suggesting that the stress response involves the differential expression of not annotated regions. Notably, we observed significant post-race increases of reads that correspond to repeats, especially the intergenic and intronic L1 and L2 transposable elements. We also observed increased expression of the antisense strands compared to the sense strands in intronic and regulatory regions (1 kb up- and downstream) of the genes, suggesting that antisense transcription could be one of the main mechanisms for transposon regulation in the horse under stress conditions. We identified a large number of transcripts corresponding to intergenic and intronic regions putatively associated with new transcriptional elements. Gene expression and pathway analysis allowed us to identify several biological processes and molecular functions that may be involved with exercise-induced stress. Ontology clustering reflected mechanisms that are already known to be stress activated (e.g., chemokine-type cytokines, Toll-like receptors, and kinases), as well as "nucleic acid binding" and "signal transduction activity" functions. There was also a general and transient decrease in the global rates of protein synthesis, which would be expected after strenuous global stress. In sum, our network analysis points toward the involvement of specific gene clusters in equine exercise-induced stress, including those involved in inflammation, cell signaling, and immune interactions.

Figure 1. (A) Basal (T1) and race (T2) sample reads map to different genomic regions.

The majority of the reads map to known genes (CDS, 3′ UTR and 5′ UTR), while a large fraction maps to non-coding regions (introns, intergenic regions, and the 1-kb regions up- and downstream of genes). Comparison between T1 and T2 show a transcriptional shift from coding to non-coding predicted regions. (B) Expression density was calculated as number of reads normalized by the lengths of each genomic region. (C) Fraction of bases covered in the different genomic regions. (D) Fraction of reads that map to the sense (light) and antisense (dark) strands in each genomic region. In the intergenic region, the fraction was calculated using the number of reads from the plus and minus strands.

Figure 2. Venn diagram showing the number of splice sites identified in the T1 and T2 samples.

A) Splicing sites confirmed by previously reported annotation of horse genes. B) Novel splicing sites.

Figure 3. Histogram showing intergenic and intronic fragment lengths (A and B) and distribution of expression (C and D panel).

Figure 4. Network 1 and 2 results from IPA analysis.

Genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge. All connections are supported by at least one reference from the literature or canonical information stored in the Ingenuity knowledge base. The intensity of the node color indicates the degree of up-regulation (red) or down-regulation (green).

Figure 5. Network 1 and 2 results from IPA analysis.

Genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge. All connections are supported by at least one reference from the literature or canonical information stored in the Ingenuity knowledge base. The intensity of the node color indicates the degree of up-regulation (red) or down-regulation (green).