Noncatalytic Assembly of Ribonuclease III with Double-Stranded RNA

Błaszczyk, Jarosław; Gan, Jianhua; Tropea, Joseph E.; Court, Donald L.; Waugh, David S.; Ji, Xinhua

doi:10.1016/j.str.2004.02.004

Cited by 117 publications

(170 citation statements)

References 42 publications

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“…These studies also indicated that a subclass of dsRBDs prefers stem-loops over A-form RNA helices [33,35]. In some structures, a sequence-specific contact has been observed between a main-chain carbonyl of the β1-β2 loop and an amino group of a guanosine in the minor groove [32,34,36,37]. In addition, a large variety of contacts have been described between helix α1 and both regular minorgrooves [32,34,36,37] and apical loop structures [33,35,38].…”

Section: Introductionmentioning

confidence: 92%

“…The structures of different dsRBDs have been determined uncovering a mixed α/β fold with a conserved αβββα topology in which the two α-helices are packed against the three-stranded anti-parallel β-sheet [30,31]. In addition, structures of dsRBDs have been determined in complex with dsRNA, predominantly with non-natural RNA duplexes [32][33][34][35][36][37][38], revealing the canonical mode of dsRNA recognition by dsRBDs. Molecular recognition is made via three regions of interaction: helix α1 and the loop between β1 and β2 contact dsRNA minor grooves at one turn of interval whereas the short loop between β3 and α2 together with the N-terminal part of helix α2 contact the dsRNA phosphate backbone across the major groove.…”

Section: Introductionmentioning

confidence: 99%

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Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

2012

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Adenosine deaminases that act on RNA (ADAR) catalyze adenosine to inosine (A-to-I) editing in double-stranded RNA (dsRNA) substrates. Inosine is read as guanosine by the translation machinery; therefore A-to-I editing events in coding sequences may result in recoding genetic information. Whereas vertebrates have two catalytically active enzymes, namely ADAR1 and ADAR2, Drosophila has a single ADAR protein (dADAR) related to ADAR2. The structural determinants controlling substrate recognition and editing of a specific adenosine within dsRNA substrates are only partially understood. Here, we report the solution structure of the N-terminal dsRNA binding domain (dsRBD) of dADAR and use NMR chemical shift perturbations to identify the protein surface involved in RNA binding. Additionally, we show that Drosophila ADAR edits the R/G site in the mammalian GluR-2 pre-mRNA which is naturally modified by both ADAR1 and ADAR2. We then constructed a model showing how dADAR dsRBD1 binds to the GluR-2 R/G stem-loop. This model revealed that most side chains interacting with the RNA sugar-phosphate backbone need only small displacement to adapt for dsRNA binding and are thus ready to bind to their dsRNA target. It also predicts that dADAR dsRBD1 would bind to dsRNA with less sequence specificity than dsRBDs of ADAR2. Altogether, this study gives new insights into dsRNA substrate recognition by Drosophila ADAR.

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Section: Introductionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

2012

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“…Previous observations that dsRBMs bind to perfect dsRNA, in a sequence-independent manner (Schreiber et al, 1989;Ryter and Schultz, 1998;Ramos et al, 2000;Blaszczyk et al, 2004), have suggested that the dsRBMs of ADAR2 may recognize all of their RNA targets in a similar manner. Consistent with this idea, ADAR2 has been shown to nonspecifically deaminate adenosine moieties in a wide range of synthetic RNA duplexes Liu et al, 1999;Lehmann and Bass, 2000;Cho et al, 2003;Dawson et al, 2004).…”

Section: Discussionmentioning

confidence: 93%

“…The lack of obvious sequence requirements for the binding of dsRBM-containing proteins led to the initial conclusion that these motifs only confer general dsRNA-binding affinity with little sequence preference, yet most of the data regarding this hypothesis focused upon model RNA substrates that contained a perfect RNA duplex (Ryter and Schultz, 1998;Ramos et al, 2000;Blaszczyk et al, 2004). By contrast, more recent studies using naturally occurring dsRNAs, formed by intramolecular base pairing between imperfect, inverted repeats, have indicated that dsRBMs have unique functional properties based upon intrinsic binding preferences and affinities (Spanggord et al, 2002;Stephens et al, 2004;Stefl et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

“…The dsRBM is a highly conserved 65-75 amino acid (aa) domain, present in many eukaryotic proteins with diverse cellular functions, that forms an ␣1-␤1-␤2-␤3-␣2 topology in which the two ␣-helices are packed along a face of a three-stranded antiparallel ␤-sheet, with most of the potential RNA-binding residues exposed on one surface (FierroMonti and Mathews, 2000;Tian et al, 2004). Structural analyses of dsRBMs complexed to short, synthetic RNA duplexes have suggested that dsRBM binding is independent of RNA sequence, because the majority of dsRBM-RNA interactions involve direct contact with the 2Ј-hydroxyl groups of the ribose sugars and direct or water-mediated contacts with nonbridging oxygen residues of the phosphodiester backbone (Ryter and Schultz, 1998;Ramos et al, 2000;Blaszczyk et al, 2004). The two dsRBMs of ADAR2, with Ͼ80% amino acid sequence similarity, adopt the same fold as all other members of the dsRBM family, although the two domains differ slightly from one another in the orientation of ␣1 helix relative to the other secondary structural elements (Stefl et al, 2006).…”

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confidence: 99%

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Substrate-dependent Contribution of Double-stranded RNA-binding Motifs to ADAR2 Function

Wells

Emeson

2006

MBoC

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ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine (A-to-I). ADAR2 contains two tandem double-stranded RNA-binding motifs (dsRBMs) that are not only important for efficient editing of RNA substrates but also necessary for localizing ADAR2 to nucleoli. The sequence and structural similarity of these motifs have raised questions regarding the role(s) that each dsRBM plays in ADAR2 function. Here, we demonstrate that the dsRBMs of ADAR2 differ in both their ability to modulate subnuclear localization as well as to promote site-selective A-to-I conversion. Surprisingly, dsRBM1 contributes to editing activity in a substrate-dependent manner, indicating that dsRBMs recognize distinct structural determinants in each RNA substrate. Although dsRBM2 is essential for the editing of all substrates examined, a point mutation in this motif affects editing for only a subset of RNAs, suggesting that dsRBM2 uses unique sets of amino acid(s) for functional interactions with different RNA targets. The dsRBMs of ADAR2 are interchangeable for subnuclear targeting, yet such motif alterations do not support site-selective editing, indicating that the unique binding preferences of each dsRBM differentially contribute to their pleiotropic function. INTRODUCTIONThe conversion of adenosine to inosine (A-to-I) by RNA editing is mediated by a family of double-stranded RNA (dsRNA)-binding proteins referred to as adenosine deaminases that act on RNA (ADARs) (Bass et al., 1997). Two mammalian enzymes in this family, ADAR1 and ADAR2, can catalyze the hydrolytic deamination of multiple sites in synthetic dsRNAs, or mediate the site-specific modification of naturally occurring viral and cellular mRNA transcripts containing extended duplex regions formed by the presence of imperfect inverted repeats (Rueter and Emeson, 1998;Bass, 2002). The most extensively studied substrates of Ato-I conversion are transcripts encoding ionotropic glutamate receptor (GluR) subunits and the 2C-subtype of the serotonin receptor (5-HT 2C R), in which editing leads to nonsynonymous codon changes that generate channels with altered electrophysiologic and ion permeation properties (Seeburg and Hartner, 2003) and receptors with decreased G protein-coupling efficiency (Burns et al., 1997;Niswender et al., 1999;Berg et al., 2001). A-to-I modifications have also been described in nontranslated RNAs and noncoding regions of mRNA transcripts, suggesting that such RNA modifications affect other aspects of RNA function, including splicing, translation efficiency, nuclear retention, and transcript stability (Rueter et al., 1999;Athanasiadis et al., 2004;Blow et al., 2004;Kim et al., 2004;Levanon et al., 2004;DeCerbo and Carmichael, 2005;Prasanth et al., 2005).ADAR2 displays a modular organization with two tandem dsRNA-binding motifs (dsRBMs) connected by a flexible linker and a conserved adenosine deaminase domain toward the carboxy terminus, for which the structures have r...

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How do ADARs bind RNA? New protein‐RNA structures illuminate substrate recognition by the RNA editing ADARs

Thomas

Beal

2017

BioEssays

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Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the genome. What determines which adenosines in an mRNA are subject to this modification reaction? The answer lies in an understanding of the mechanism and substrate recognition properties of adenosine deaminases that act on RNA (ADARs). Our recent publication of x-ray crystal structures of the human ADAR2 deaminase domain bound to RNA editing substrates shed considerable light on how the catalytic domains of these enzymes bind RNA and promote adenosine deamination. Here we review in detail the deaminase domain-RNA contact surfaces and present models of how full length ADARs, bearing double stranded RNA-binding domains (dsRBDs) and deaminase domains, could process naturally occurring substrate RNAs.

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Noncatalytic Assembly of Ribonuclease III with Double-Stranded RNA

Cited by 117 publications

References 42 publications

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

Substrate-dependent Contribution of Double-stranded RNA-binding Motifs to ADAR2 Function

How do ADARs bind RNA? New protein‐RNA structures illuminate substrate recognition by the RNA editing ADARs

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