The diverse functions of the ADAR enzymes: Editing and editing-independent effects on gene expression

Abstract

The adenosine deaminase acting on RNA (ADAR) family of enzymes consists of three members (ADAR1-3). These enzymes are responsible for the deamination of adenosine into inosine in double-stranded RNA, one of the most common RNA modifications in mammals. Inosine is treated as guanosine by the cellular machineries and the functional rewriting of the genetic code has profound consequences for the cell. The ADAR enzymes are RNA binding proteins that also can affect the transcriptome through editing independent activity. This thesis focuses on investigating editing independent functions of the ADAR proteins, with a special focus on the catalytically inactive ADAR3.

We studied the expression of ADAR3 throughout embryonic brain development and observed that ADAR3 is only expressed in a subpopulation of in vitro differentiated primary cortical neurons, suggesting a specific neuronal function. We generated a transgenic cell line that exogenously expresses ADAR3 and applied methods such as co-immunoprecipitation, RNA sequencing and proteomics in order to further understand ADAR3’s function. We revealed the protein interactome of ADAR3 and found links to translation and RNA stability. We showed that ADAR3 associates with polysomes and inhibits translation, and our results suggest that ADAR3 binds to target mRNAs and stabilizes them in non-productive polysome complexes. Moreover, ADAR3 expression changed the levels and stability of several mRNAs involved in neuronal differentiation and impeded neurofilament outgrowth in in vitro differentiation experiments. We propose that ADAR3 negatively regulates neuronal differentiation and that it does so by regulating mRNA stability and translation.

In another study, we investigate the impact of ADAR editing and RNA binding on miRNA biogenesis. We observed that the editing levels of the majority of mature miRNAs expressed from the miR-379-410 cluster was lower than the editing levels detected in the precursor transcript. One exception was miR-376b-3p, which exhibited higher levels of editing in its mature form. We analyzed the biogenesis of miR-376-3p and showed that its maturation is negatively regulated by ADAR2 in an editing independent manner, while ADAR1 promotes its biogenesis. The edited version of miR-376b-3p targeted a different set of mRNAs in comparison to its unedited counterpart. One of these targets was ABAT, a protein responsible for GABA catabolism. Expression of the edited miR-376b-3p led to downregulation of ABAT and an increase in intracellular GABA. These studies showed that ADARs modulate the expression of miR-376b-3p through both catalytic and non-catalytic mechanisms, and that this modulation has the potential to regulate GABAergic signaling in the brain.

In the third study, we investigated editing dependent and independent roles of ADAR1 on alternative splicing. We sequenced the transcriptome of four human cell lines with perturbed ADAR1 expression levels. Our results showed that the majority of the alternative splicing events regulated by ADAR1 are editing independent. In addition, we showed that ADAR1 affects the splicing of transcripts that encode splicing factors, and that some of the differential splicing events that are affected by perturbations in ADAR1 expression are also affected upon alteration in splicing factor expression. These results suggest that ADAR1 can indirectly regulate splicing in a global manner by regulating the alternative splicing of specific splicing factors.

Keywords: RNA editing, ADAR, Alternative splicing, miRNA processing, neuronal differentiaton.