Physiological and pathological perspectives in the biology of APOBEC deaminases

The thesis is focus on RNA editing mediated by two AID/APOBEC family members. The aim of my work was the investigation of possible novel factors that regulate hAPOBEC1 expression or cofactors which help the deaminase to exert its activity. First, I characterised cellular models for their proliferation and clonogenic activities as well as cell cycle distribution evaluating a combinatorial effect of hAPOBEC1 and RBM47 which lead to a decrease in cell growth. I investigated the role of RNA editing beyond the lipid transport by high-throughput sequencing which provided me information regarding new deamination events, RNA stability, and also a differential gene expression in presence or absence of the editosome components. By Differential expression analysis, I got a list of genes that are differentially expressed in clones with hAPOBEC1 and RBM47 which need to be analysed for their biological meaning. From the mRNA-seq I got a consistent list of putative edited sites even though some of them were validated with no success. Moreover, I applied a genetic library screen to activate a high number of genes in cells expressing RBM47 to evaluate an eventual up-regulation of APOBEC1 and find factors which trigger its expression. The cells in which editing happened have been selected thanks to a specific fluorescent reporter containing ApoB target. The results have still to be analysed. The second aim of my project was to study APOBEC3A regulation, by chemical and genetic screenings, through the development of a specific sensitive reporter system to detect APOBEC3A-mediated RNA editing. In this work I presented the design of an artificial fluorescent reporter containing a target of APOBEC3A like SDHB or DDOST properly built to produce a stop codon in the middle of the target and optimised for the levels of editing. I checked its specificity for APOBEC3A and not for other APOBEC proteins like APOBEC1 and APOBEC3B. This let me also detected a novel putative editing site mediated by APOBEC3A by Sanger sequencing. Moreover, I designed another fluorescent reporter system able to evaluate APOBEC3A RNA editing by fluorescent microscopy. I created stable cell lines expressing all the lentiviral reporter plasmids to further investigate induction of endogenous APOBEC3A and its regulation. In a future perspective the dual fluorescent reporter could be a useful tool to identify novel RNA editing targets upon the application of an activation library screen.