Effects of ALS-associated 5'tiRNAGly-GCC on the transcriptomic and proteomic profile of primary neurons in vitro

tRNA-derived stress-induced RNAs (tiRNAs) are a new class of small non-coding RNA that have emerged as important regulators of cellular stress responses. tiRNAs are derived from specific tRNA cleavage by the stressinduced ribonuclease angiogenin (ANG). Loss-of-function mutations in the ANG gene are linked to amyotrophic lateral sclerosis (ALS), and elevated levels of specific tiRNAs were recently identified in ALS patient serum samples. However, the biological role of tiRNA production in neuronal stress responses and neurodegeneration remains largely unknown. Here, we investigated the genome-wide regulation of neuronal stress responses by a specific tiRNA, 5'tiRNAGly-GCC, which we found to be upregulated in primary neurons exposed to ALS-relevant stresses and in the spinal cord of three ALS mouse models. Whole-transcript RNA sequencing and label-free mass spectrometry on primary neurons transfected with a synthetic mimic of 5'tiRNAGly-GCC revealed predominantly downregulated RNA and protein levels, with more pronounced changes in the proteome. Over half of the downregulated mRNAs contained predicted 5'tiRNAGly-GCC binding sites, indicating that this tiRNA may silence target genes via complementary binding. On the proteome level, we observed reduction in proteins involved in translation initiation and ribosome assembly, pointing to inhibitory effects on translation. Together, these findings suggest that 5'tiRNAGly-GCC is an ALS-associated tiRNA that functions to fine-tune gene expression and supress protein synthesis as part of an ANG-induced neuronal stress response.