Sex-based eRNA expression and function in ischemic stroke.

Enhancer-derived RNAs (eRNAs) are a new class of long noncoding RNA that have roles in modulating enhancer-mediated gene transcription, which ultimately influences phenotypic outcomes. We recently published the first study mapping genome-wide eRNA expression in the male mouse cortex during ischemic stroke and identified 77 eRNAs that were significantly altered following a 1 h middle cerebral artery occlusion (MCAO) and 6 h of reperfusion, as compared to sham controls. Knockdown of one such stroke-induced eRNA - eRNA_06347 - resulted in significantly larger infarcts, demonstrating a role for eRNA_06347 in modulating the post-stroke pathophysiology in males. In the current study, we applied quantitative real-time PCR to evaluate whether the 77 eRNAs identified in the male cortex also show altered expression in the post-stroke female cortex. Using age-matched and time-matched female mice, we found that only a subset of the 77 eRNAs were detected in the post-stroke female cortex. Of these, only a small fraction showed similar temporal expression characteristics as males, including eRNA_06347 which was highly induced in both sexes. Knockdown of eRNA_06347 in the female cortex resulted in significantly increased infarct volumes that were closely matched to those in males, indicating that eRNA_06347 modulates the post-stroke pathophysiology similarly in males and females. This suggests a common underlying role for eRNA_06347 in the two sexes. Overall, this is the first study to evaluate eRNA expression and perturbation in the female cortex during stroke, and present a comparative analysis between males and females. Our findings show that eRNAs have sex-dependent and sex-independent expression patterns that may be of significance to the pathophysiological responses to stroke in the two sexes.

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.