Mass-spectrometry based screening revealing proteins bound to FMR1 mRNA

Short nucleotide repeats are robustly distributed in human genome and contribute to pathogenesis of multiple neurodegenerative disorders such as Fragile X-associated Tremor/Ataxia Syndrome (FXTAS). Pathogenesis of FXTAS is driven by premutation status of CGG repeats expansion (CGGexp) in the 5’UTR region of fragile X messenger ribonucleoprotein 1 (FMR1) gene, when CGG triplet varies between 55-200. One of the proposed molecular mechanism involved in disease progression is repeats associated non-AUG (RAN) translation, which results in the production of toxic aggregation-prone protein called FMRpolyG. Mechanistic insights of RAN translation remain elusive, therefore we aimed to identify novel RAN translation modifiers. In order to identify factors involved in FMRpolyG synthesis we applied RNA-tagging system combined with mass spectrometry (MS) based protein identification to elucidate pool of proteins natively bound to FMR1 transcript harboring CGGexp mimicking FXTAS premutation status. As a control RNAm we used construct enriched with G and C nucleotides (named GCrich RNA) to mimic GC content in investigated FMR1 mRNA. Overall, performed MS-based screening revealed novel proteins which bind specifically to 5’UTR of FMR1 with CGGexp in cellulo.