Laboratory for Stroke Research and Noncoding RNA Biology, JFK Neuroscience Institute, HackensackMeridian Health JFK Medical Center, 65 James Street, Edison, NJ, 08820, USA.
Mol Neurobiol. 2019 Feb;56(2):1035-1043. doi: 10.1007/s12035-018-1147-0. Epub 2018 Jun 3.
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.
基因表达在脑缺血中已经成为多年来的研究热点。利用基于探针的高通量方法（如微阵列）的研究极大地促进了我们现有的知识，但缺乏详细分析转录组的能力。全基因组 RNA 测序（RNA-seq）能够全面检查转录组的属性，如链特异性、选择性剪接、选择性转录起始/终止位点和序列组成，从而提供基因表达的非常详细的描述。利用这一能力，我们利用 RNA-seq 对成年小鼠皮质在再灌注 6、12 或 24 小时后的短暂局灶性缺血进行了深入的全基因组蛋白编码转录组评估。与假手术对照组相比，我们在再灌注 6 小时时总共鉴定出 1007 个转录本，在再灌注 12 小时时鉴定出 1878 个转录本，在再灌注 24 小时时鉴定出 1618 个转录本。在异构体水平分辨率下，我们从 23 个基因中鉴定出了 23 个产生新 mRNA 异构体的剪接变体。对于一部分基因，我们检测到再灌注时间点依赖性剪接异构体转换，表明这些基因的表达和/或功能转换。最后，在所有三个再灌注时间点上，我们发现了 286 个基因，每个基因都有多个不同的、同时表达和差异表达的异构体，这些异构体是通过不同的转录起始/终止位点产生的。其中，109 个基因的 165 个异构体是新的 mRNA。总的来说，我们的数据以空前的深度揭示了大脑皮质的蛋白编码转录组，为中风相关基因转录和转录组织的灵活性和复杂性提供了一些新的见解。
