Structural analysis of human ADAR2-RNA complexes by X-ray crystallography

Abstract

Adenosine deaminases acting on RNAs (ADARs) are a class of RNA editing enzymes found in metazoa that catalyze the hydrolytic deamination of adenosine to inosine in duplexed RNA. Inosine is a nucleotide that can base pair with cytidine, therefore, inosine is interpreted by cellular processes as guanosine. ADARs are functionally important in RNA recoding events, RNA structure modulation, innate immunity, and can be harnessed for therapeutically-driven base editing to treat genetic disorders. Guide RNAs (gRNAs) bearing various modifications can be used to recruit ADARs to edit sites of interest in a process called site-directed RNA editing (SDRE). To help advance the rational design of gRNAs for therapeutics, characterizing the structure-to-activity relationship of ADARs' recognition and binding of substrate duplex RNA at atomic resolution is critical. In this chapter, we describe the process of determining the structure of human ADAR2 bound to duplex RNA using X-ray crystallography. Solid phase synthesis of 8-azanebularine-modified RNAs and purification for binding and crystallographic studies are described. The overexpression and purification of ADARs and assembly of the protein-RNA complex are detailed. Lastly, methods for crystallizing ADAR-RNA complexes and X-ray structure determination and data refinement strategies are outlined.

Keywords: ADAR; Adenosine; Adenosine deaminase; Inosine; RNA editing.

Figure 1.

Domain maps of hADAR2 constructs. hADAR2-FL: full length human ADAR2 (aa1–701), hADAR2-R2D: human ADAR2 (aa215–701), hADAR2-D: human ADAR2 (aa299–701).

Figure 2.

SDS-PAGE gel of fractionated hADAR2-R2D-EQ after the first Ni-NTA stained with SYPRO orange. Fractions F5 – F16 were pooled. hADAR2-R2D-EQ’s expected molecular weight (MW) is ~55 kDa.

Figure 3.

Ni-NTA 2 with hADAR2-R2D-EQ post-TEV reaction. The gels were stained with Coomassie Blue. (Left) Gel after the wash step where hADAR2 flows through the column and fractions F2 through F5 were pooled. (Right) Elution of TEV protease from the nickel column with high imidazole concentration.

Figure 4.

SDS-PAGE of TEV protease fractions post-Ni-NTA. Fractions F1-F11 were pooled. The gel was stained with SYPRO orange, and the molecular weight of TEV is ~37 kDa.

Figure 5.

EMSA gel of ADAR2-R2D E488Q binding to the RNA duplex shown with 8ZN and ³²P incorporated in the top strand. Gel shift assays were performed 0 to 256 nM protein added.

Figure 6.

Native PAGE hybridization check gels of RNA duplexes. Gels were run on a 20% polyacrylamide TBE gel. Each single strand and duplex (50 picomoles) were loaded in each individual lane. Gels were stained for 30 min with SYBR^™ Gold. (A) Hybridization check gel for a 19 mer duplex for crystallography. (B) Hybridization for an 18 mer duplex for crystallography.

Figure 7.

X-ray crystal structures of hADAR-RNA complexes reveal important interactions. (A) A high-resolution structure of hADAR2-R2D E488Q bound to duplex RNA revealed an alpha helix (light green) bearing conserved residues that make important contacts across the protein dimer interface (e.g. D503). (B) The D503A mutant shows a substantially reduced rate of deamination for the 5-HT2cR mRNA D site (Thuy-Boun et al. 2020).