Mechanistic insights into editing-site specificity PNAS PLUS of ADARs Ashani Kuttan and Brenda L. Bass1 Department of Biochemistry, University of Utah, Salt Lake City, UT 84112 Edited by Joan A. Steitz, Howard Hughes Medical Institute, New Haven, CT, and approved October 9, 2012 (received for review July 20, 2012) Adenosine deaminases that act on RNA (ADARs) deaminate base-paired dsRNA of 50 bp or more, whereas adenosines in adenosines in dsRNA to produce inosines. ADARs are essential in shorter dsRNA or in dsRNA containing mismatches, bulges, and mammals and are particularly important in the nervous system. loops are edited more selectively (9–11). ADARs’ dsRBMs are Altered levels of adenosine-to-inosine (A-to-I) editing are observed believed to play a large role in selectivity (12). in several diseases. The extent to which an adenosine is edited The extent of A-to-I editing at a particular site depends on depends on sequence context. Human ADAR2 (hADAR2) has 5′ sequence context, and these rules are referred to as “preferences” and 3′ neighbor preferences, but which amino acids mediate these (11, 13). Human ADAR1 (hADAR1) and human ADAR2 preferences, and by what mechanism, is unknown. We performed (hADAR2) have a 5′ nearest-neighbor preference of U > A > C > a screen in yeast to identify mutations in the hADAR2 catalytic Ganda3′ nearest-neighbor preference of G > C ∼A > UandG> domain that allow editing of an adenosine within a disfavored C > U ∼A, respectively (14). Truncated forms of hADAR1 and triplet. Binding affinity, catalytic rate, base flipping, and preferen- hADAR2 comprising only the catalytic domain have the same 5′ ces were monitored to understand the effects of the mutations on preference as the full-length proteins and similar but distinct 3′ ADAR reactivity. Our data provide information on the amino acids preferences (G > C > A > UandC∼G ∼A > U, respectively) that affect preferences and point to a conserved loop as being of (14). The 3′ preferences of the truncated forms indicate that the ′ key importance. Unexpectedly, our data suggest that hADAR2’s dsRBMs play a role in the 3 neighbor preference, as is consistent preferences derive from differential base flipping rather than from with NMR solution structures of mammalian ADAR2 dsRBMs direct recognition of neighboring bases. Our studies set the stage bound to the R/G hairpin of GRIA2 that show a hydrogen bond ′ BIOCHEMISTRY for understanding the basis of altered editing levels in disease and from S258 in the second dsRBM to the amino group of the 3 G for developing therapeutic reagents. (15). In another study, when the deaminase domains of hADAR1 and hADAR2 are switched, substrate specificity of the chimeric 2-aminopurine | RNA editing protein tracks with its deaminase domain (16). These studies suggest that preferences derive mainly from the catalytic domain, but which amino acids in the catalytic domain mediate preferences denosine deaminases that act on RNA (ADARs) target is not known. Adouble-stranded regions of precursor mRNAs (pre-mRNAs), To identify the amino acids that mediate preferences, we per- noncoding RNAs, and viral RNAs, deaminating adenosines to – formed a screen for mutations within the hADAR2 catalytic do- create inosines (1 3). Inosine is recognized as guanosine; thus, main that allow editing of an adenosine in a poor sequence adenosine-to-inosine (A-to-I) editing in a pre-mRNA can alter context. Collectively, the hADAR2 variants we identified point to codons and splice-forms, leading to multiple protein isoforms a conserved loop near the active site as important for preferences. from a single gene. ADARs also alter microRNA and endogenous ’ – Unexpectedly, our data suggest that hADAR2 s preferences de- siRNA biogenesis and targeting (2 4). A-to-I editing of viral rive from differential base flipping rather than from direct rec- RNAs can reduce virus growth as well as enhance it (5). ognition of the neighboring bases. These studies offer insight into ADARs are found in most metazoans, and often more than one the correlation of altered editing levels with disease and set the ADAR exists in an organism. For example, there are three mam- stage for developing therapeutic reagents. malian ADAR genes: ADAR1, ADAR2,andADAR3, and each has two or three N-terminal dsRNA-binding motifs (dsRBMs) and Results a highly conserved C-terminal deaminase domain. ADAR1 and Screen Identifies Residues in the hADAR2 Catalytic Domain That ADAR2 are active deaminases, but enzymatic activity has not been Affect Preferences. For both hADAR1 and hADAR2, nearest- observed with ADAR3 (6, 7). neighbor preferences derive mainly from the catalytic domain Two of the most studied ADAR substrates are the pre-mRNAs (14, 16). A crystal structure of the catalytic domain of hADAR2 of glutamate receptor, ionotropic, AMPA 2 (GRIA2) and the 5- has been solved (17); therefore, to facilitate our analysis, we HT2C serotonin receptor. GRIA2 pre-mRNA has two editing sites, focused on this enzyme. We adapted a previously reported one that recodes glutamine into arginine (Q/R), and another that screen in Saccharomyces cerevisiae (18) to identify mutations in recodes an arginine into glycine (R/G). Aberrant A-to-I editing is the hADAR2 catalytic domain that allow editing of an adenosine correlated with several diseases (8). For example, underediting of in the context of a disfavored triplet, GAC, where the underline the Q/R site of GRIA2 pre-mRNA is implicated in amyotrophic indicates the targeted adenosine. lateral sclerosis, overediting of the R/G site is observed in epilepsy The screen relied on a hairpin-reporter that was introduced patients, and an increase in editing of the 5-HT2C serotonin re- into the chromosome of the haploid yeast strain, W303α, under ceptor pre-mRNA is observed in patients with depression and in suicide victims. In addition, the locus for dyschromatosis sym- metrica hereditaria (DSH), a pigmentary genodermatosis, maps to the ADAR1 gene. The mechanistic basis for altered levels of editing in various diseases is entirely unclear. Author contributions: A.K. and B.L.B. designed research; A.K. performed research; A.K. ADARs specifically edit certain adenosines over others, and the and B.L.B. analyzed data; and A.K. and B.L.B. wrote the paper. extent of editing also varies. There are two determinants of The authors declare no conflict of interest. specificity: selectivity and preferences. The fraction of sites edited This article is a PNAS Direct Submission. in a dsRNA, referred to as “selectivity,” depends on its length and 1To whom correspondence should be addressed. E-mail: [email protected]. whether it contains mismatches, bulges, and internal loops (3). In This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vitro studies show that nonselective editing occurs in completely 1073/pnas.1212548109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1212548109 PNAS Early Edition | 1of10 Downloaded by guest on October 4, 2021 ADADAR editing Stop 5’- - 3’ = Green cells Secretion signal mRNA B UGI(Tryptophan) UGA (Stop) A A G A C hp GAC 5’-CCGUUUG CUGGGUGGAUA UAUACC U 3’-GGCAGGC GAUCCACCUAU AUAUGG C C C G C A C U I G (Tryptophan) UAG (Stop) A A G U GAC yeast strain UAG yeast strain 5’-CCGUUU GGUGGGUGGAUA UAUACCA U hp UAG Assay with Wildtype hADAR2 3’-GGCAGA CCAUCCACCUAU AUAUGGU C C C G G E WT E488Q N597K V493A N613K A589V G336D S599T T490A hp GAC +++ ++++++ hp UAG ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ FGT490A A589 E488Q V493A N597K N613K E488 ADAR2 LRTK I ESGEGT IPV PNFSVNWTVGD SAI EV I NAT G336 N597 T490 ADAR2 LRTK I ESGEGT IPV PNFSVNWTVGD TAI EV I NAT S599 ADAR2 LRTK I ESGEGT IPV PNFSVNWTVGD ATIEVI NAT N613 V493 ADAR2 LRTK I ESGEGT IPV PNFSVNWTVGD TGLEV INAT ADAR2 LRTK I ESGEGT IPV PNFGI NWT I GD TELEVVNSL ADAR2 LRTK I ESGEGT IPV PNFRVNWTVGD QGLKVI NAT ADAR2 LRTK I ESGEGT IPV PNFSVNWTVGD QGLEI I NAT ADAR2 LRTK I ESGEGT IPV PNFGI NWRRND DSFEVINAM ADAR3 LRTK I ESGEGTVPV PPFSMNWVVGS AD LEI I NAT ADAR1 LRTKVENGEGT IPV KETSVNWCLADGY DLEILDGT hADAR2: 480-493 hADAR2: 596-615 Fig. 1. Mutants that edit the disfavored GAC hairpin were identified from a screen in yeast. (A) Schematic of the hairpin-reporter used in the screen. The hairpin, in red, is an ADAR substrate with the target adenosine in context of a stop codon that must be edited for expression of the downstream α-galac- tosidase reporter. (B and C) Sequences of the GAC (B) and UAG (C) hairpins (hp) with the target adenosine within a disfavored and favored triplet, re- spectively. (D) Control experiments showing CM −URA plates with yeast colonies that have a GAC (Left)orUAG (Right) hairpin-reporter integrated into a chromosome and transformed with WT hADAR2. (E) Mutants identified from the screen, listed from left to right in terms of decreasing green intensity of yeast colonies, an indication of decreasing in vivo editing efficiency. Green intensity of yeast colonies with the control UAG hairpin-reporter is indicated also. “++++” indicates that yeast colonies started turning green in ≤4d;“+++” and “++” indicate 5–7 d, and “+” indicates low levels of editing taking 2–5wkto turn faint green. (F) Mutated residues (yellow sticks) mapped onto the crystal structure of the catalytic domain of hADAR2 (Protein Data Bank ID code 1ZY7) (17) with Zn (pink sphere), IP6 (orange and red stick), and modeled in AMP (pink stick). (G) Alignment of hADAR1, hADAR2, hADAR3, and ADAR2 from different species. Mutants that were characterized further are indicated. The highly conserved loop that includes two β-strands and comprises 14 residues is highlighted in yellow. the control of a constitutive promoter, ADH1. The hairpin that introduction of WT hADAR2 into strains containing the (shown in red in Fig.