A multiplex platform for small RNA sequencing elucidates multifaceted tRNA stress response and translational regulation

Small RNAs include tRNA, snRNA, micro-RNA, tRNA fragments and others that constitute >90% of RNA copy numbers in a human cell and perform many essential functions. Popular small RNA-seq strategies limit the insights into coordinated small RNA response to cellular stress. Small RNA-seq also lacks multiplexing capabilities. Here, we report a multiplex small RNA-seq library preparation method (MSR-seq) to investigate cellular small RNA and mRNA response to heat shock, hydrogen peroxide, and arsenite stress. Comparing stress-induced changes of total cellular RNA and polysome-associated RNA, we identify a coordinated tRNA response that involves polysome-specific tRNA abundance and synergistic N3-methylcytosine (m3C) tRNA modification. Combining tRNA and mRNA response to stress we reveal a new mechanism of stress-induced down-regulation in translational elongation. We also find that native tRNA molecules lacking several modifications are biased reservoirs for the biogenesis of tRNA fragments. Our results demonstrate the importance of simultaneous investigation of small RNAs and their modifications in response to varying biological conditions. Overall design: This study contains 64 samples divided between the following experimentes: CMC treatment, demethylase treatment, input titrations, stressed tRNA, stressed tRNA from polysomes, stressed mRNA, and stressed mRNA from polysomes. 4 samples are associated with CMC project, two replicates of RNA with and without N-cyclohexyl-N'-ß-(4-methylmorpholinium) ethylcarbodiimide (CMC) treatment. 6 samples are associated with demethylase treatment, three replicates of RNA with and without AlkB demethylase treatment. 6 samples are assoicated with input titrations, two replicates for each of 10ng 100ng and 1000ng input amounts. 12 samples are associated with stressed tRNA, three replicates for each of no stress, heat stress, H2O2 stress, and AsO2. 12 samples are assoicated with stressed tRNA from polysomes, three replicates from four conditions as before. 12 samples are assoicated with stressed mRNA, three replicates from four treatments as before. 12 samples are assoicated with stress mRNA from polysomes, three replicates from four treatments as before.