APPLICATION OF ESCHERICHIA COLI AND 293T MODEL SYSTEMS TO STUDY THE HUMANPOLYRIBONUCLEOTIDE PHOSPHORYLASE

RNA metabolism refers to all processes of the life of a ribonucleic acid, including processing and degradation, which are governed by enzymes, among which is polynucleotide phosphorylase (PNPase). This remarkable enzyme is found across many prokaryotic and eukaryotic organisms, and it can work either as a 3’–5’ exoribonuclease for RNA degradation or as a polymerase for RNA synthesis. In humans, PNPase (hPNPase) resides within the mitochondria, although evidence of its presence in other cellular compartments is reported. Severe conditions can arise from genetic variants in the hPNPase-encoding gene, termed PNPT1. In this work, the generation of model systems in Escherichia coli and in 293T human cells was exploited to test the effect of pathological mutations associated with different diseases. The integration of data derived from both models has helped to assess that pathological mutations cause defects in protein stability and expression to varying extents. In E. coli, activation of the SOS response was observed upon expression of hPNPase from the chromosome, which is instead abolished when pathogenic variants are present. This unique phenotype allowed the generation of a biosensor strain expressing GFP coupled to the activity of hPNPase in E. coli that can be used for screening campaigns of chemicals restoring the activity of the mutated variants. In 293T cells, two variants have been introduced by genome editing, allowing, on one hand, the validation of the results obtained in E. coli, and on the other, the implementation of a valuable pathogenic cell model in human cells that could help elucidating cellular defects due to PNPT1 mutations.