Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.
Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.
['Bhattarai|Sunil|S|', 'Aly|Ahmed|A|', 'Garcia|Kristy|K|', 'Ruiz|Diandra|D|', 'Pontarelli|Fabrizio|F|', 'Dharap|Ashutosh|A|http://orcid.org/0000-0001-5223-3197']
D017398:Alternative Splicing D000818:Animals D002545:Brain Ischemia D002544:Cerebral Infarction D020869:Gene Expression Profiling D018740:Genetic Heterogeneity D059014:High-Throughput Nucleotide Sequencing D008297:Male D008810:Mice, Inbred C57BL D020033:Protein Isoforms D017423:Sequence Analysis, RNA D020521:Stroke D059467:Transcriptome
Ischemia Gene expression RNA-sequencing Mouse Cerebral cortex