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

Abstract

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.

A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1

“…To assess the nucleic acid binding properties of dsRBD constructs, we produced RNA substrates by in vitro transcription with T7 polymerase, namely a dsRNA duplex of 24 bp as previously described (36), and the GluR-2 R/G editing site stem-loop (GluR-2 LSL), a typical ADAR substrate used previously for structural studies with ADAR2 (41). RNAs were purified by anion-exchange HPLC under denaturing conditions as previously described (42). DNA templates were purchased from Microsynth AG.…”

Conformational dynamics at microsecond timescale in the RNA-binding regions of dsRNA-binding domains

“…To investigate the timescale of motions present in the proteins, NMR spin relaxation data (R 1 , R 2 and [ 1 H]-15 N NOE) was analysed for 69 non-overlapping peaks out of 96 assigned peaks for TRBP2-dsRBD1 (Paithankar et al, 2018) and for 58 such peaks out of 74 assigned peaks for dADAR-dsRBD1 (Barraud et al, 2012) ( Figure S1). The analysis of the data using the extended model-free approach Szabo, 1982b, 1982a;Clore et al, 1990) allowed the extraction of dynamic parameters at the ps-ns timescale.…”

“…Diffusion tensor values for various diffusion models -sphere, spheroid, and ellipsoid -were calculated using the program quadric diffusion (Lee et al, 1997) for data recorded at both the magnetic fields. Solution structure of TRBP2-dsRBD1 (Paithankar et al, 2018) and dADAR-dsRBD1 (Barraud et al, 2012) was used to optimize the diffusion tensor parameters. These diffusion tensor parameters were then used to calculate the model-free parameters from the ten model-free models and the best model for each residue was selected based on Akaike's Information Criteria.…”

“…In addition to canonical α 1 -β 1 -β 2 -β 3 -α 2 secondary fold, an additional N-terminal helix α 0 was recently detected at the N terminus of dsRBD1 of TRBP2 (isoform1 of TRBP) (Masliah et al, 2018;Paithankar et al, 2018), the function of which remains to be understood. The second model protein used for the current investigation is the ADAR protein, known to catalyze the conversion of Adenosine to Inosine (A → I), which is critical for regulating gene expression (Bass, 2007;Barraud et al, 2012;Savva, Rieder and Reenan, 2012). Human ADAR2 protein is reported to interact with dsRNAs in a sequencespecific manner (Stephens, Haudenschild and Beal, 2004;Stefl et al, 2010), while Drosophila ADAR-dsRBD1 is reported to have less sequence-specificity (Barraud et al, 2012).…”

“…The second model protein used for the current investigation is the ADAR protein, known to catalyze the conversion of Adenosine to Inosine (A → I), which is critical for regulating gene expression (Bass, 2007;Barraud et al, 2012;Savva, Rieder and Reenan, 2012). Human ADAR2 protein is reported to interact with dsRNAs in a sequencespecific manner (Stephens, Haudenschild and Beal, 2004;Stefl et al, 2010), while Drosophila ADAR-dsRBD1 is reported to have less sequence-specificity (Barraud et al, 2012). While both the proteins contain multiple dsRBDs, this study focusses on the dsRBD1 of these proteins to allow understanding of the intrinsic dynamics of single dsRBD (keeping any other possible domain-domain interaction aside).…”