Identification of Dominant Transcripts in Oxidative Stress Response by a Full-Length Transcriptome Analysis

Area of Science:

• Molecular Biology
• Genomics
• Cellular Stress Response

Background:

• The body employs multilayered regulatory mechanisms, including the KEAP1-NRF2 system, to respond to environmental stress by altering cytoprotective enzyme/protein expression.
• Short-read RNA sequencing (RNA-Seq) presents limitations in accurately interpreting cytoprotective gene induction at the transcript level, causing confusion regarding effectiveness.
• Understanding precise isoform usage is crucial for a clear picture of the cellular stress response.

Purpose of the Study:

• To precisely delineate isoform usage in the cellular stress response using full-length transcriptome profiling.
• To compare the effectiveness of long-read sequencing (Iso-Seq) versus short-read sequencing (RNA-Seq) in analyzing transcript isoform abundance.
• To investigate the regulation of transcript usage in NRF2 target genes under electrophilic stress.

Main Methods:

• Conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) on lymphoblastoid cells from three volunteers.
• Analyzed cells under both normal and electrophilic stress-induced conditions.
• Determined first exon usage in KEAP1 and NFE2L2 (encoding NRF2) and examined isoform usage changes in NRF2 target genes.

Main Results:

• Identified transcript diversity in KEAP1 and NFE2L2, indicating complex gene regulation.
• Discovered dominant isoforms in the majority of NRF2 target genes under stress conditions.
• Observed significant discrepancies between Iso-Seq and RNA-Seq in determining transcript isoform abundance, with RNA-Seq potentially being misleading.

Conclusions:

• Transcript usage is tightly regulated to produce functional proteins during electrophilic stress.
• Long-read transcriptome sequencing (Iso-Seq) offers significant advantages over short-read RNA-Seq for accurate transcriptomic analysis.
• Iso-Seq provides a more precise understanding of gene expression regulation in cellular stress responses.