Adar1 restricts line-1 retrotransposition

Adenosine deaminases acting on RNA (ADARs) are enzymes that catalyze the hydrolytic deamination of Adenosine (A) to Inosine (I) (AàI) in double-stranded RNA (dsRNA) substrates. This process is called RNA editing. In literature there are many studies describing ADAR1 as regulator of the replication of different viruses, exerting either a proviral or antiviral effect, depending on the type of virus and the host. Our laboratory and others demonstrated that ADAR1 exerts a proviral effect on HIV-1 replication by both mechanisms editing-dependent and editing-independent. Indeed, in HIV-1 ADAR1 enhances the viral proteins expression through a mechanism editing-independent and stimulates the release of progeny virions that display even a higher infectious capacity, through a mechanism editing-dependent. We also demonstrated that ADAR1 interacts with the p55-Gag protein, the principal structural component of the immature virions, and probably this interaction is sufficient for ADAR1 incorporation into the viral particles. Starting from these results, the initial aim of my PhD was to identify the putative ADAR1- interactors during HIV-1 expression, with the final goal of bringing to light proteins that could stimulate ADAR1 proviral activity. The initial step of this work was to isolate the native ADAR1 ribonucleoprotein complex (RNP) in HIV-1 expressing cells using a dual tag affinity purification system followed by the identification of the RNP components by mass spectrometry analysis. Using this approach we isolated fourteen non-ribosomal proteins as putative interactors of ADAR1, most of which are novel. Strikingly, a good fraction of the identified ADAR1-interactors (TOP1, PABPC1, NCL, hnRNP L and HSPA1A) were previously reported to be components of the Long Interspersed Element 1 (LINE-1 or L1) RNPs and some of them regulate the retrotransposition mechanism. L1 elements are autonomous retrotransposons that may reintegrate itself in the human genome by a round of retrotransposition. This result suggests a possible functional link between ADAR1 and L1 retrotransposons. To confirm this hypothesis, we first validated the association between ADAR1 and these interactors shared with the L1 retrotransposons, by performing immunoprecipitation (IP) experiments. Next, we tested whether ADAR1 could play a role in the regulation of LINE-1 retrotransposition. To this aim, we employed two widely used retrotransposition assays. In particular, we studied the effect of knock-down of ADAR1 expression on LINE-1 retrotransposition efficiency. To reach this goal, we used specific plasmids expressing short-harping RNA (shRNA) directed against ADAR1 mRNA. By using this approach we demonstrated that the reduction of ADAR1 expression causes an increase in L1 retrotransposition efficiency, thus strongly suggesting a novel function of ADAR1 as repressor of L1 retrotransposon activity. To further confirm these results, we tested the effect of ADAR1 over-expression on L1 activity and as expected, we showed that increased amount of ADAR1 protein causes a decrease in L1 retrotransposition. Moreover, we assayed whether the RNA editing activity of ADAR1 is required for the inhibition of LINE-1 retrotransposition. To this aim, we performed specific experiments using a mutant form of ADAR1 that lacks the catalytic domain and we analysed the effect of this mutant in comparison to the wt ADAR1 on L1 retrotransposition. The results of these experiments suggest that the editing activity of ADAR1 is not required or only marginally involved in the inhibition of LINE-1 retrotransposition. In support of this data, we have not found AàI editing events in the sequence of the ectopically expressed L1 RNAs in cells over-expressing ADAR1. Finally, to investigate the mechanism by which ADAR1 control the L1 retrotransposition, we performed IP experiments to assay whether ADAR1 can interact with some components, RNA or proteins of the LINE-1 ribonucleoparticles. The result of these experiments demonstrated that ADAR1 interacts with the L1 RNA and also with the retrotransposon protein ORF1p. Based on the results obtained, our hypothesis is that ADAR1 inhibits L1 retrotransposition by binding the L1 RNP complex thus impairing its activity. Moreover we also tested if ADAR1 could have a role in the regulation of Alu elements retrotransposition. We employed a specific retrotransposition assay and we demonstrated that the knock-down of ADAR1 expression causes an increase of Alu retrotransposition activity. In conclusion, all of these data demonstrate a novel role of ADAR1 as inhibitor of LINE-1 and Alu retrotransposition.