Our research delves into the genetic underpinnings of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), with a specific focus on the prevalent C9orf72 gene mutation. This mutation involves the expansion of a hexanucleotide repeat sequence - GGGGCC - transcribed in both sense (G4C2)n and antisense (C4G2)n directions, leading to the formation of nuclear RNA foci. A pivotal finding in our recent study is the discovery of an interaction between antisense C4G2 repeat RNA and phenylalanine-tRNA synthetase (FARS), resulting in diminished charging of phenylalanine-tRNA and consequent impact on the expression of proteins rich in phenylalanine content. Through bioinformatic analysis, we revealed that the majority of proteins high in phenylalanine content localize to membranes, with the endoplasmic reticulum being particularly prominent. Utilizing techniques such as immunocytochemistry analysis and subcellular fractionation coupled with western blots, we observed notable downregulation of these proteins in cells derived from C9orf72 patients. Additionally, we investigated stress responses, notably the induction of the starvation response in the presence of the repeats. Furthermore, we developed two novel methods, RNA-RNA proximity ligation assay (PLA) and fluorescence in situ hybridization (FISH) coupled with flow cytometry, offering enhanced sensitivity and ease of analysis compared to traditional FISH techniques. Our study highlights the significance of aminoacyl-tRNA synthetases and phenylalanine-rich proteins in C9orf72 ALS/FTD, suggesting their potential role in disease development. These innovative methods provide valuable tools for probing C9orf72 mutation-derived RNAs, paving the way for novel research avenues and therapeutic strategies.1Črnigoj M.M., Čerček U et al. Nat. Commun. 2023, PMID: 37717009