Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method

Abstract

Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5' side of the modification site. This 5' binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure-activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5' binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure-function relationships in ADAR reactions.

Figure 1.

5′ RNA binding loop of hADAR2. (A) Alignment of sequences of the ADAR2 5′ binding loop from different organisms. The conserved residues in the RNA binding loop are colored in blue with residues in the variable region black. (B) Crystal structure of hADAR2 deaminase domain bound to dsRNA substrate (27). The 5′ RNA binding loop is highlighted in blue.

Figure 2.

Design and characterization of an RNA editing reporter for single yeast cell analysis. (A) Schematic of reporter mRNA. (B) BDF2-derived sequence used as substrate with edited A indicated by an arrow (30). (C) Full sequence of reporter near editing site with the encoded protein sequence shown. (D) Images of cells expressing the yeGFP reporter and ADAR deaminase domains detected by fluorescence microscopy. Scale bar: 100 μm. (E) Fluorescence measurements of cell suspensions. F/F0 is the ratio of sample fluorescence divided by negative control (inactive mutant) fluorescence. Error bar indicates SD, n ≥ 3. (F) Change of fluorescence of cells expressing hADAR2-D with time. Error bar indicates SD, n ≥ 3. (G) FACScan analysis of cells expressing inactive E396A mutant and wild-type hADAR2-D.

Figure 3.

(A) Sat-FACS-Seq workflow for defining structure–function relationships in the 5′ binding loop of hADAR2. (B) Fluorescence activated cell sorting. Top left: Cells expressing the yeGFP reporter and hADAR2-D E396A were used to define background fluorescence and above-background fluorescence categories. Top right: Cells expressing the yeGFP reporter and hADAR2-D loop library were sorted into different categories based on different levels of fluorescence. Bottom: Parameters summarized corresponding to each sorting channel.

Figure 4.

Normalized average fluorescence corresponding to each of the 20 common amino acids at 19 positions in the hADAR2 5′ binding loop. The star means stop codon.

Figure 5.

Functional determinants for the 5′ binding loop of hADAR2-D revealed by the screening. (A) Logo plot for residues 454–479 summarizing the frequency of each amino acid at each position in the most fluorescent category (R5). Positions where the wild-type residue is highly preferred are labeled with red stars. (B) Structural evidence showing a network of interactions among the essential residues and the substrate RNA (27).