Abstract
Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine within double-stranded regions of RNA, resulting in increased transcriptomic diversity, as well as protection of cellular double-stranded RNA (dsRNA) from silencing and improper immune activation. The presence of dsRNA-binding domains (dsRBDs) in all ADARs suggests these domains are important for substrate recognition; however, the role of dsRBDs in vivo remains largely unknown. Herein, our studies indicate the Caenorhabditis elegans ADAR enzyme, ADR-2, has low affinity for dsRNA, but interacts with ADR-1, an editing-deficient member of the ADAR family, which has a 100-fold higher affinity for dsRNA. ADR-1 uses one dsRBD to physically interact with ADR-2 and a second dsRBD to bind to dsRNAs, thereby tethering ADR-2 to substrates. ADR-2 interacts with >1200 transcripts in vivo, and ADR-1 is required for 80% of these interactions. Our results identify a novel mode of substrate recognition for ADAR enzymes and indicate that protein–protein interactions can guide substrate recognition for RNA editors.
Highlights
Diverse enzymes catalyze modification of nucleotides in all types of nucleic acids present in the cell
By analyzing WT and mutant ADAR proteins in vitro as well as in vivo, we demonstrate that ADR-1 and ADR-2 have a direct protein–protein interaction that involves the second dsRNA-binding domains (dsRBDs) of ADR-1
Using both in vitro doublestranded RNA (dsRNA) binding and transcriptome-wide examination of ADR-2 association with cellular RNAs, we found that ADR-1 has >100-fold greater affinity for dsRNA compared to ADR-2, and stable interaction of ADR-2 with most target messenger RNAs (mRNAs) in vivo requires ADR-1
Summary
Diverse enzymes catalyze modification of nucleotides in all types of nucleic acids present in the cell. While transfer RNAs (tRNAs) are the most extensively modified molecules in the cell [1], recent technological developments have enabled the detection of several types of modifications in eukaryotic messenger RNAs (mRNAs) [2,3]. Deamination of adenosine (A) results in inosine (I), which has similar base-pairing properties as guanosine. Due to these differences in base pairing, A-to-I editing can alter the amino acid encoded by a codon, modify splice sites and affect the interaction of the RNA molecule with itself or other RNAs, such as microRNAs [5]. A-to-I editing in long double-stranded regions has been reported to prevent silencing of host RNAs and improper activation of the immune response by self-RNAs [6,7,8]
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