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 KEAP1 and NFE2L2 (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.

中文翻译：

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通过全长转录组分析鉴定氧化应激反应中的主要转录本

我们的身体通过包括KEAP1-NRF2系统在内的多层调节机制，通过改变一系列细胞保护酶/蛋白质的表达水平来应对环境压力。尽管NRF2上调了许多细胞保护性基因的表达，但短读RNA测序（RNA-Seq）仍存在基本局限性，导致在转录水平上解释细胞保护性基因诱导的有效性时产生了困惑。为了精确描述应激反应中亚型的使用，我们进行了独立的全长转录组谱分析（亚型测序； Iso-Seq），分析了三名志愿者在正常和亲电应激条件下的淋巴母细胞。我们首先确定了KEAP1和NFE2L2中的第一个外显子用法（编码NRF2），并发现转录本多样性的存在。然后，我们检查了NRF2靶基因在压力条件下的同工型使用情况的变化，并确定了在大多数NRF2靶基因中主要表达的几种同工型。通过Iso-Seq分析确定的同工型的表达水平与通过短读RNA-Seq确定的同工型存在显着差异。后者可能会导致笔录数量过多而产生误导。这些结果支持严格控制转录物的使用以在亲电子胁迫下产生功能性蛋白质。我们目前的研究强烈认为，通过长时间阅读转录组测序可以实现重要的益处。