Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
Department of Medical Biochemistry, Graduate School of Medicine, Tohoku University, Sendai, Japan.
Mol Cell Biol. 2021 Jan 25;41(2). doi: 10.1128/MCB.00472-20.
Our body responds to environmental stress by changing the expression levels of a series of cytoprotective enzymes/proteins through multilayered regulatory mechanisms, including the KEAP1-NRF2 system. While NRF2 upregulates the expression of many cytoprotective genes, there are fundamental limitations in short-read RNA sequencing (RNA-Seq), resulting in confusion regarding interpreting the effectiveness of cytoprotective gene induction at the transcript level. To precisely delineate isoform usage in the stress response, we conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) analyses of lymphoblastoid cells from three volunteers under normal and electrophilic stress-induced conditions. We first determined the first exon usage in and (encoding NRF2) and found the presence of transcript diversity. We then examined changes in isoform usage of NRF2 target genes under stress conditions and identified a few isoforms dominantly expressed in the majority of NRF2 target genes. The expression levels of isoforms determined by Iso-Seq analyses showed striking differences from those determined by short-read RNA-Seq; the latter could be misleading concerning the abundance of transcripts. These results support that transcript usage is tightly regulated to produce functional proteins under electrophilic stress. Our present study strongly argues that there are important benefits that can be achieved by long-read transcriptome sequencing.
我们的身体通过多层次的调节机制，包括 KEAP1-NRF2 系统，来改变一系列细胞保护酶/蛋白的表达水平，以应对环境压力。虽然 NRF2 上调了许多细胞保护基因的表达，但短读长 RNA 测序（RNA-Seq）存在根本的局限性，导致在转录水平上解读细胞保护基因诱导的效果时存在混淆。为了精确描绘应激反应中的异构体使用情况，我们对来自三个志愿者的淋巴母细胞系在正常和亲电应激诱导条件下进行了独立的全长转录组谱分析（异构体测序；Iso-Seq）。我们首先确定了 和 （编码 NRF2）的第一外显子使用情况，并发现了转录多样性的存在。然后，我们检测了应激条件下 NRF2 靶基因异构体使用情况的变化，并鉴定出了少数在大多数 NRF2 靶基因中表达占主导地位的异构体。Iso-Seq 分析确定的异构体表达水平与短读长 RNA-Seq 确定的表达水平存在显著差异；后者可能会对转录本的丰度产生误导。这些结果表明，在亲电应激下，转录物的使用受到严格调控以产生功能性蛋白。我们的研究强烈支持长读长转录组测序可以带来重要的益处。
