Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia

• Published in: Molecular neurobiology Vol. 56; no. 2; pp. 1035 - 1043
• Main Authors: Bhattarai, Sunil, Aly, Ahmed, Garcia, Kristy, Ruiz, Diandra, Pontarelli, Fabrizio, Dharap, Ashutosh
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.02.2019; Springer Nature B.V
• Subjects: Alternative splicing; Alternative Splicing - genetics; Animals; Biomedical and Life Sciences; Biomedicine; Brain Ischemia - genetics; Cell Biology; Cerebral cortex; Cerebral Infarction - genetics; DNA microarrays; Gene expression; Gene Expression Profiling - methods; Genetic Heterogeneity; Genomes; High-Throughput Nucleotide Sequencing - methods; Ischemia; Isoforms; Male; Mice, Inbred C57BL; Neurobiology; Neurology; Neurosciences; Period protein; Protein Isoforms - metabolism; Proteins; Reperfusion; Sequence Analysis, RNA; Stroke - genetics; Transcription; Transcriptome - genetics; RNA-sequencing; Cerebral cortex; Mouse; Ischemia; Gene expression
• ISSN: 0893-7648; 1559-1182; 1559-1182
• DOI: 10.1007/s12035-018-1147-0

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.