The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1 - PubMed (original) (raw)

The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1

A Herbert et al. Proc Natl Acad Sci U S A. 2001.

Abstract

RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. Here the role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 is examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5' to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11-15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited.

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Figures

Figure 1

ADAR1 constructs used in these studies. All constructs had a N-terminal FLAG tag and a C-terminal HIS tag. Both the long (ADAR1) and short (M246) forms of ADAR1 are shown. Sites at which mutations were made are indicated by an asterisk and by the position of the mutation. A _Swa_I site was created at position 750, allowing the in-frame deletion of the three DRBMs by using the naturally occurring _Swa_I site at position 485 to produce ΔR123. The nuclear export signal (NES) (residues 128–137; A.H., H. Knaut, A.R., and J. Nickerson, unpublished work), Z-DNA-binding domains (Zα and Zβ), and the DRBM (R1, R2, R3) are labeled.

Figure 2

The position of a mismatch determines which adenosine is edited. Four dsRNA editing substrates were examined that incorporated the sequence AUAUA in the top strand, as shown in the lower part of the figure. Each substrate differs in the position of a cytosine mismatch. M29a has a cytosine mismatch opposite the second A, M29b opposite the third A, and M29c opposite the first A. Editing was compared with the M29D substrate that had no mismatches. The upper part of the figure shows a sequencing gel where sites of A to G editing are boxed and marked with an asterisk. These sites are also marked with an asterisk in the sequences shown in the lower part of the figure. In these stem–loop structures, capital letters are used for Watson–Crick base pairs, lower case for mismatched or unpaired bases, the solid lines connect adjacent nucleotides, and the top line starts with the 5′ end of the sequence.

Figure 3

The M7G editing substrate is edited on both strands of the dsRNA stem. Editing is observed even when ADAR1 DRBMs are absent or mutated. M7G was created from the wild-type GluR-B pre-mRNA RG site by replacing the residues that are underlined. Editing was observed at five sites in M7G (numbered 1–5, 5′ to 3′). The same numbering is used for the wild-type substrate. The ability of different ADAR1 constructs to edit the sites in M7G was compared (the band marked as R was used as a reference for quantitation). M246 is the short form of ADAR1 and serves as a positive control. Mqaa has the catalytic domain of M246 inactivated by mutation and serves as a negative control. K744 contains only the catalytic domain of ADAR1. ΔR123 has all three DRBMs in M246 deleted. MR123 has the following mutations that inactivate RNA binding: K504E, K615E and K723E.

Figure 4

The relative position of editing sites in a dsRNA is not fixed. M7L has editing sites E2 and E3 separated by 11 bp compared with 15 bp between E2 and E5 in M7G (Fig. 3.). This change in spacing places E3 in M7L and E5 in M7G on different sides of the dsRNA helix with respect to E2. Comparison of Fig. 4 with Fig. 3 also shows that editing of E2, which is placed 4 bp from the end of the duplex in M7L, is diminished when compared with M7G, which has E2 6 bp from the end. Editing of E1, which is 3 bp from the end of the dsRNA stem, is also greatly reduced in M7L.

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The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1 - PubMed (original) (raw)