Proteins binding to duplexed RNA: one motif, multiple functions

Fierro-Monti, Ivo; Mathews, Michael B.

doi:10.1016/s0968-0004(00)01580-2

Cited by 230 publications

(222 citation statements)

References 49 publications

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“…The dsRBD is a ~65-75 amino acids domain with specific binding capacity for dsRNA, which is found in many eukaryotic and prokaryotic proteins presenting a large variety of functions [26][27][28][29]. 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].…”

Section: Introductionmentioning

confidence: 99%

“…Even if most of these contacts are not sequence-specific, it has been proposed earlier that helix α1 could help achieve substrate specific recognition via the modulation of its contacts with apical RNA loop structures [33]. Nevertheless, the most common idea about dsRBDs is that they recognize the A-form helix of dsRNA in a sequence-independent manner [26,29,32]. Indeed, the majority of dsRBD-RNA interaction involve contacts with the 2′-hydroxyl groups of the ribose sugar rings and with non-bridging oxygen of the phosphodiester backbone.…”

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: 99%

Section: Introductionmentioning

confidence: 99%

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

2012

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“…A majority of the proteins involved in the recognition of duplex RNA contain multiple copies of a highly conserved 65-to 75-aa dsRNAbinding motif, providing a molecular mechanism to facilitate the interaction of these proteins with their dsRNA targets (Fierro-Monti and Mathews, 2000;Doyle and Jantsch, 2002;Carlson et al, 2003;Tian et al, 2004). ADAR2 is a dsRNA-specific adenosine deaminase that also contains two tandem copies of this highly conserved motif (Tian et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

“…Because deletion or even subtle point mutations of ADAR2 dsRBMs could result in structural alterations that cause ADAR2 mislocalization, we also examined the subcellular localization of eYFP when expressed as a fusion protein with either dsRBM1 (aa 74 -147) or dsRBM2 (aa 231-301) alone ( Figure 2C); the precise borders for each motif were based upon amino acid sequence homology to other dsRBMcontaining proteins and the recently resolved NMR structure for these domains in ADAR2 (Fierro-Monti and Mathews, 2000;Stefl et al, 2006). Both eYFP-dsRBM1 and eYFP-dsRBM2 were localized to the cytoplasm and nucleus, presumably because of the absence of a nuclear localization signal and the passive diffusion of in these small fusion proteins (ϳ40 kDa) through the nuclear pore (Suntharalingam and Wente, 2003); however, eYFP-dsRBM1 was localized to nucleoli significantly better than eYFP-dsRBM2 (p Ͻ 0.001; Figure 2C), further confirming a nonequivalent role for these highly …”

Section: The Role Of Dsrbms In the Nucleolar Localization Of Adar2mentioning

confidence: 99%

“…The relative nucleolar fluorescence for the ⌬dsRBM1 mutant was significantly less than for the fusion protein lacking dsRBM2 (p Ͻ 0.02), suggesting a greater role for dsRBM1 in the subnuclear compartmentalization of ADAR2. Independent substitutions of K 127 and K 281 decreased relative nucleolar fluorescence to the same extent (K127A and K281A); however, the magnitude of this effect was less than that observed for the dsRBM deletions ( Figure 2), suggesting that additional contacts between the dsRBMs and nucleolar binding site(s) are required for normal nucleolar localization.Because deletion or even subtle point mutations of ADAR2 dsRBMs could result in structural alterations that cause ADAR2 mislocalization, we also examined the subcellular localization of eYFP when expressed as a fusion protein with either dsRBM1 (aa 74 -147) or dsRBM2 (aa 231-301) alone ( Figure 2C); the precise borders for each motif were based upon amino acid sequence homology to other dsRBMcontaining proteins and the recently resolved NMR structure for these domains in ADAR2 (Fierro-Monti and Mathews, 2000;Stefl et al, 2006). Both eYFP-dsRBM1 and eYFP-dsRBM2 were localized to the cytoplasm and nucleus, presumably because of the absence of a nuclear localization signal and the passive diffusion of in these small fusion proteins (ϳ40 kDa) through the nuclear pore (Suntharalingam and Wente, 2003); however, eYFP-dsRBM1 was localized to nucleoli significantly better than eYFP-dsRBM2 (p Ͻ 0.001; Figure 2C), further confirming a nonequivalent role for these highly …”

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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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Determination of the structure of the RNA complex of a double-stranded RNA-binding domain fromDrosophila Staufen protein

Ramos¹,

Bayer²,

Varani³

1999

Biopolymers

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We have determined using NMR the structure of the complex between the third double‐stranded RNA‐binding domain (dsRBD3) of Drosophila Staufen protein and a RNA stem‐loop with optimal binding properties in vitro. This work was designed to understand how dsRBD proteins bind RNA and to investigate the role of Staufen dsRBDs in the localization of maternal RNAs during early embryonic development. The structure determination was challenging, because of weak, nonsequence specific binding and residual conformational flexibility at the RNA–protein interface. In order to overcome the problems originated by the weak interaction, we used both new and more traditional approaches to obtain distance and orientation information for the protein and RNA components of the complex. The resulting structure allowed the verification of aspects of RNA recognition by dsRBDs matching the information obtained by a related crystallographic study. We were also able to generate new observations that are likely to be relevant to dsRBD–RNA binding and to the physiological role of Staufen protein.©2001 John Wiley & Sons, Inc. Biopoly( Nucleic Acid Sci) 52: 181–196, 1999/2000

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Proteins binding to duplexed RNA: one motif, multiple functions

Cited by 230 publications

References 49 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

Determination of the structure of the RNA complex of a double-stranded RNA-binding domain fromDrosophila Staufen protein

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