A new strategy to increase RNA editing at the Q/R site of GluA2 AMPA receptor subunits by targeting alternative splicing patterns of ADAR2

Abstract

Background: The GluA2 subunit of AMPA receptors (AMPARs) undergoes RNA editing at a specific base mediated by the enzyme ADAR2, changing the coded amino acid from a glutamine to arginine at the so-called Q/R site, which is critical for regulating calcium permeability. ADAR2 exists as multiple alternatively-spliced variants within mammalian cells with differing editing efficiency.

New method: In this study, phosphorodiamidate morpholino oligomers (PMOs) were used to increase Q/R site editing, by affecting the alternative splicing of ADAR2.

Results: PMOs targeting the ADAR2 pre-mRNA transcript successfully induced alternative splicing around the AluJ cassette leading to expression of a more active isoform with increased editing of the GluA2 subunit compared to control.

Comparison with existing method(s): Previously PMOs have been used to disrupt RNA editing via steric hindrance of the GluA2 RNA duplex. In contrast we report PMOs that can increase the expression of more catalytically active variants of ADAR2, leading to enhanced GluA2 Q/R RNA editing.

Conclusions: Using PMOs to increase Q/R site editing is presented here as a validated method that would allow investigation of downstream cellular processes implicated in altered ADAR2 activity.

Keywords: ADAR2; AMPARs; Amyotrophic lateral sclerosis; Antisense oligonucleotides; Neurodegeneration.