Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions

Abstract

Transcripts encoding ADAR1, a double-stranded, RNA-specific adenosine deaminase involved in the adenosine-to-inosine (A-to-I) editing of mammalian RNAs, can be alternatively spliced to produce an interferon-inducible protein isoform (p150) that is up-regulated in both cell culture and in vivo model systems in response to pathogen or interferon stimulation. In contrast to other tissues, p150 is expressed at extremely low levels in the brain and it is unclear what role, if any, this isoform may play in the innate immune response of the central nervous system (CNS) or whether the extent of editing for RNA substrates critical for CNS function is affected by its induction. To investigate the expression of ADAR1 isoforms in response to viral infection and subsequent alterations in A-to-I editing profiles for endogenous ADAR targets, we used a neuro-tropic strain of reovirus to infect neonatal mice and quantify A-to-I editing in discrete brain regions using a multiplexed, high-throughput sequencing strategy. While intracranial injection of reovirus resulted in a widespread increase in the expression of ADAR1 (p150) in multiple brain regions and peripheral organs, significant changes in site-specific A-to-I conversion were quite limited, suggesting that steady-state levels of p150 expression are not a primary determinant for modulating the extent of editing for numerous ADAR targets in vivo.

Comparative analysis of A-to-I editing in human and non-human primate brains reveals conserved patterns and context-dependent regulation of RNA editing

“…Editing profiles were determined by high-throughput multiplexed transcript (HTMTA) as described in [23] [12]. Reactions amplifying each respective substrate transcript were performed in parallel using one of 24 unique barcoded primer sets allowing for 24 sample multiplexing.…”

Altered intrathalamic GABAA neurotransmission in a mouse model of a human genetic absence epilepsy syndrome

“…We dissected VB / nRT regions from brain slices as described above and quantified Gabra3 RNA editing using a high-throughput multiplexed transcript analysis as described previously (Hood et al, 2014). …”

Mutations underlying Episodic Ataxia type-1 antagonize Kv1.1 RNA editing

“…The extent of editing was quantified by high-throughput sequence analysis as described by Hood et al . 33 and used to calculate the rate of editing ( Fig. 1b ).…”

“…Reactions were terminated by the addition of TRIzol (Ambion) and RNA was extracted according to the manufacturer’s protocol. RNA was reverse-transcribed with random primers using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and the extent of RNA editing was quantified by high-throughput multiplexed sequence analysis as described previously 33 . The editing rate was calculated as the fmol RNA converted to the edited isoform divided by the duration of the reaction.…”