Rapid dynamics of stress-induced cholinergic-targeted small RNAs in human embryonic stem cells

Human embryonic stem cells (hESCs) are a powerful model for exploring cellular stress responses. Here, we report potential contributions of microRNAs (miRs) and the recently re-discovered transfer RNA-derived fragments (tRFs) in hESCs stress responses. MiRs/tRFs may both block the expression of mRNAs, interact with numerous RNA-binding proteins and suppress entire cellular pathways in a tissue-specific and condition-dependent manner. Moreover, miRs are known post-transcriptional regulators of hESCs differentiation over several days or weeks. However, the dynamics of miRs and tRFs in short-term stress regulation is unknown. To induce a severe short-term stress, we isolated RNA 1, 3, 6, 24 and 48 hours following depletion from the hESCs media of basic fibroblast growth factor (bFGF), an essential component for hESCs’ survival as non-differentiated stem cells. Small RNA-sequencing provided an overview of miRs/tRFs dynamics within the first 24h after bFGF removal. Briefly, bFGF-depleted hESCs presented significant differential expression (DE) in the levels of 186 tRFs and 235 miRs. Of those, 6/36 and 76/68 miRs/tRFs were DE by 1h and 6h post-culturing, indicating an accelerating role for tRFs in hESCs with response time to these stressful conditions. Furthermore, 33%/25% and 39.5%/14.7% of those miRs/tRFs target at least 5 cholinergic-related transcripts each. These include the acetylcholinesterase (AChE)-targeting and inflammation-controlling hsa-miR-132-3p, the pain-regulating hsa-miR-21-3p, the ribosomes production-inducer tRF-22-WB8647O52 and the miR/tRF-18-HR0VX6D2. Those predictably target mRNAs involved in cell adherence, fatty acid metabolism, cellular stress responses, adherence and differentiation. Small ncRNAs thus emerge as important regulatory elements of the short-term response of hESCs to acute stress.