ADAR2, short for adenosine deaminases acting on RNA, is an RNA editing enzyme that converts specific adenosines to inosines (A-to-I) in certain pre-mRNAs. A-to-I editing leads frequently to amino acid substitutions in the encoded proteins. ADAR2 has been best characterized as selective editing enzyme, in the nervous system.In addition, recent studies identified non-coding RNAs such as miRNAs as ADAR2 targets. ADAR2 may also participate in A-to-I editing of repetitive RNA sequences in intronic and untranslated sequences of messenger RNAs.
Of all ADAR2-mediated edits, a single one in the pre-mRNA of the AMPA receptor subunit GluA2 is essential for survival in mice. Hence, early postnatal death of mice lacking ADAR2 is averted when the critical edit is engineered into both GluA2 encoding Gria2 alleles (Gria2R/R). Thus, Adar2-/-/Gria2R/R mice (ADAR2 ko mice) appear normal and have a normal life span. However, a comprehensive analysis of the Adar2-/-/Gria2R/R phenotype covering all organ systems has been lacking until now. In this study the Adar2-/-/Gria2R/R mouse line was compared to Gria2R/R mice as controls, to study the phenotypic consequences of loss of all ADAR2-mediated edits except the critical one in GluA2. The extended phenotype analysis in the German Mouse Clinic (GMC) identified significant changes caused by the absence of ADAR2 in behavior, hearing ability, allergy parameters and transcript profiles of brain. These findings most likely suggest the requirement of ADAR2 for the editing of additional transcripts other than the previously identified major target GluA2.
In the allergy screen of the GMC a statistically significant difference in total IgE plasma levels in Adar2-/-/Gria2R/R females was identified. RNA editing by activation-induced deaminase (Aid) has been reported to be crucial for the class-switching mechanism of distinct immunoglobulins. Changes of total IgE plasma levels in ADAR2 ko mice might suggest potential ADAR2 targets which alter IgE plasma levels. It will be of interest to analyze the allergy and IgE phenotype of ADAR2 ko mice under conditions of allergic sensitization and airway challenge using a model allergen.
The hearing ability of Adar2-/-/Gria2R/R mice was assessed by a click box test producing a short 20 kHz sound and assessing the Preyer’s reflex (ducking the head, closing eyes and flicking ears backwards). This test identified a reduced or absent Preyer’s reflex in male mutant mice. This mutant phenotype might be caused by functional deficits of the cochlea or reduction in motor recruitment or both.
In addition, prepulse inhibition (PPI) of the acoustic startle reflex (ASR) was in the GMC used to assess sensorimotor gating of Adar2-/-/Gria2R/R mice. Deficits in PPI generally are caused by the inability to filter important from unimportant information and are characteristic also for patients suffering from diseases such as schizophrenia or Alzheimer’s disease. A significant change in PPI response was observed in male ADAR2 ko mice. These alterations indicate a change in sensorimotor integration in Adar2-/-/Gria2R/R mutant mice. The 5-HT2C serotonin receptor is a known ADAR2 substrate. Changes in this receptor signaling may thus be the cause for the observed PPI deficit in the ADAR2 ko mice. Interestingly, these findings in mice are in accordance with a PPI deficit in schizophrenic patients, which show altered editing of the 5-HT2C receptor pre-mRNA in the prefrontal cortex. An altered 5-HT2C receptor function might contribute to the hearing deficit in the ADAR2 ko mice, as this receptor is expressed in both, cochlea and inferior colliculus.
The transcriptome analysis in the molecular phenotyping screen of the GMC identified pronounced changes in the expression levels of many brain transcripts, none of which is known to undergo RNA editing, so far. Several up-regulated genes are involved in neuroprotection (Cdk6, Col8a1, Dnajb6, Hdac9, and Id3) and repair processes in astrocytes (Itsn, Rgs3, and Clint1), while others are associated with synaptic trafficking. In previous analyses common targets of RNA editing were identified that are involved in nervous system functions, cell trafficking and membrane functions, and in human brain-related diseases. Similar functional annotations were also present within the dataset of regulated genes identified in the brain of ADAR2 ko mice. Therefore, these genes are potential candidate targets for ADAR2-mediated editing.
In summary, the comprehensive phenotypic analysis of the Adar2-/-/Gria2R/R mice revealed several specific mutant phenotypes. The changes found in behavior, hearing ability, allergy parameters and gene expression profiles in the brain of mutant mice now provide a rational basis for targeted studies of ADAR2 functions and for the identification of new functional ADAR2 targets.
Requirement of the RNA editing enzyme ADAR2 for normal physiology in mice, Horsch et al., J Biol Chem. 2011 May 27;286(21):18614-22. Epub 2011 Apr 5.