Gene-level RNA-seq obscures extensive transcript-level remodeling during hypoxic adaptation

Abstract

Although RNA sequencing (RNA-seq) generates transcript-resolved measurements, these data are commonly summarized to the gene level by collapsing sequencing reads into a single expression value for each gene. Because most mammalian genes express multiple transcript isoforms through alternative splicing and alternative promoter usage, this framework can obscure major changes in transcript composition and consequently misrepresent transcriptional responses. To determine the extent to which isoform-level regulation remains undetected by conventional analyses, we examined hypoxic stress responses in human vascular endothelial cells using complementary approaches that quantify differential exon usage, transcript usage, and alternative splicing across multiple durations of hypoxia. These analyses uncovered thousands of hypoxia-responsive genes not identified by gene-level approaches and revealed that the earliest response to hypoxia occurred predominantly through changes in transcript usage rather than overall expression, whereas later stages showed progressive convergence between the two levels of regulation. Notably, most alternatively spliced genes remained undetected by standard differential expression analyses throughout the time course, indicating that transcript remodeling frequently occurs independently of changes in total gene expression. Incorporating transcript-level information also substantially expanded the number of hypoxia-associated biological pathways, revealing regulatory programs not captured by conventional gene-level analyses. These findings demonstrate that gene-level RNA-seq substantially underestimates the complexity and temporal structure of hypoxic adaptation and identify transcript remodeling as a major and previously underappreciated component of the endothelial hypoxic response.