FRET Analysis of in Vivo Dimerization by RNA-editing Enzymes

Abstract

Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo-and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent. Double-stranded RNAs (dsRNAs)2 in eukaryotes are subject to a variety of processing reactions, including cleavage by the RNase III family members Drosha and Dicer in the micro RNA and small interfering RNA gene-silencing pathways and editing by members of the ADAR (adenosone deaminase that acts on RNA) enzyme family (1, 2). This latter reaction involves the hydrolytic deamination of adenosine (A) to inosine (I) within the context of dsRNA. Editing events of this type have been demonstrated in both cellular and viral transcripts and have been shown to function in altering the coding properties of the edited RNAs. For example, the life cycle of the Hepatitis ␦ virus is regulated by an editing event in the anti-genome in which a UAG stop codon is converted to a UIG tryptophan codon (3). An A to I modification is involved in the functional regulation of a growing number of cellular factors. These include the tissue-specific editing of the serotonin 5-HT2C receptor, which results in a reduction in response to serotonin agonists (4). Transcripts for subunits of the neural-specific AMPA class of glutamate-gated (GluR) ion channels undergo A to I modification at two positions, the Q/R and R/G editing sites, that affect the properties of the resulting channels (5, 6). In addition to the editing of these and other neuronal transcripts to effect codon changes, one deaminase family member, ADAR2, has been shown to autoregulate its own expression by the creation of a 3Ј-splice site (CAA to CAI) (7). Despite the identification of these editing substrates, the global role of A to I modification in higher eukaryotes remains unclear. Measurement of inosine levels in RNA isolated from rat tissue suggests a greater level of editing than indicated by known RNA substrates (8). A cloning protocol that depended upon an inosine-specific cleavage of RNA detected a large number of editing sites in non-coding regions of RNAs from Caenorhabditis elegans and humans that included sites in 5Ј-and 3Ј-untranslated regions and introns (9). Recent bioinformatic studies have suggested the presence of more than 12,000 editing sites corresponding to non-coding regions of t...

FRET-detectable interactions between the ARE binding proteins, HuR and p37AUF1

“…Alternatively, and perhaps most simply, the possibility exists that the heteromeric interactions between p37AUF1 and HuR are weaker than the respective homomeric interactions. In support of this later interpretation is the fact that E d is independent of the concentration(s) of fluors (and thus is likely not an artifact of differential expression), and the fact that FRET efficiency can under certain circumstances be correlated directly with the affinity of protein-protein interaction (Chilibeck et al 2006).…”

RNA Binding-independent Dimerization of Adenosine Deaminases Acting on RNA and Dominant Negative Effects of Nonfunctional Subunits on Dimer Functions

“…Although several publications by our group and others on the mammalian ADARs point to dimerization that is independent of RNA binding (42,43), other studies in mammalian cells and Drosophila indicate that dsRNA binding is required (40,44). Furthermore, some studies on human ADAR2 suggest that the protein exists only as a monomeric form (32,45).…”

“…Currently, it is not known whether the interplay between the monomers act cooperatively with respect to their dsRBDs. By use of fluorescence or bioluminescence resonance energy transfer, an examination of ADAR dimerization revealed that the N-terminal dsRBDs are providing much of the interface for the monomer subunits to interact in mammalian cells (43,44).…”

Down-regulation of RNA Editing in Pediatric Astrocytomas

“…Competition Experiments-ϳ7 ϫ 10 5 HEK 293T cells at 60% confluence were co-transfected with constant amounts of the GluR-B miniB13 (3 g) and of EGFP-ADAR2 (8 g) so that 50% editing of the Gln/Arg site was obtained, and an increasing amount of EGFP-ADAR1 (4,8,16, and 24 g). pEGFP (V-EGFP) was also transfected so that the total concentration of exogenous DNA transfected was equal (total DNA transfected at each experimental point was 35 g).…”