Full-length direct RNA sequencing reveals widespread RNA decay upon cellular stress

Cells respond and adapt to endogenous and exogenous stressors by activating stress response pathways that restore cellular homeostasis. The capacity of cells to respond to stress declines with age and has been associated with disease. However, the changes in cytoplasmic RNA metabolism during the cellular stress response are poorly characterized. In this work, we employ end-to-end direct RNA sequencing with nanopores, following the ligation of unique 5’ end adaptors, to assess the human transcriptome at single-molecule and -nucleotide resolution. To analyze this data, we developed NanopLen, a statistical tool to identify robust RNA length changes across conditions using linear mixed models. We find that upon cellular stress, RNAs are globally, subject to decay at their 5’ end. Stress-induced decay is coupled to translation and ribosome occupancy and can be inhibited by restoring translation initiation or preventing ribosome run-off. In contrast to models of RNA decay under normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but is independent of the poly(A) tail length. Surprisingly, we find that decaying RNAs persist in cells and are enriched in the stress granule transcriptome. Our results reveal the dynamics of post-transcriptional cell response that regulates the state of RNA upon cellular stress.