Abstract

Translation and protein synthesis are the most energy intensive processes in the cell, and as such are highly regulated during periods of stress and non-optimal growth conditions. Recent evidence has shown that amino acid starvation can affect translation by causing ribosome stalling and collisions that ultimately trigger the Ribotoxic Stress Response (RSR). The RSR results in rescue of ribosomal subunits, degradation of the nascent peptide, and mRNA decay through endonucleolytic cleavage at the stall site. However, while the RSR and resulting mRNA decay have been well-characterized mechanistically, there is currently no method to comprehensively analyze transient mRNA decay intermediates resulting from ribosome stalling. To address this limitation, we adapted a long-read sequencing approach called True End-to-end RNA Sequencing (TERAseq) to identify putative mRNA decay intermediates of the starvation-induced RSR. Using this approach, we identified an increase in potential intermediates of endonucleolytic decay and saw a starvation-dependent change in nucleotide composition around the 5’ end of identified mRNA molecules. In summary, we found that starvation caused detectable changes in mRNA features that could be uniquely identified using TERA-seq.