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 neurotropic 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.

Introduction

The complexity of signaling networks in the central nervous system (CNS) relies on the tightly-controlled regulation and continuous fine-tuning of gene expression. In addition to changes in cell-specific transcriptional activation, dynamic alterations in RNA processing events such as alternative splicing (Grabowski, 2011, Gustincich et al., 2006, Licatalosi and Darnell, 2006) and RNA editing (Balik et al., 2013, Berg et al., 2008, Sanjana et al., 2012, Schellekens et al., 2012, Tan et al., 2009) are required to achieve the precise cascade of cellular events necessary for normal neuronal function. The conversion of adenosine to inosine (A-to-I) by RNA editing is an essential cellular mechanism for diversifying the transcriptome and subsequent protein activity by introducing non-synonymous codon changes in mRNAs encoding proteins critical for nervous system activity including ligand- and voltage-gated ion channels, G-protein coupled receptors and components of the synaptic release machinery (Hood and Emeson, 2012, Hoopengardner et al., 2003, Rosenthal and Seeburg, 2012). An inosine within the open reading frame (ORF) of an mRNA is read as guanosine during translation, which can lead to specific change(s) in the predicted amino acid coding potential of the mRNA to alter the functional properties of the encoded protein product.

The conversion of A-to-I is mediated through the actions of a family of double-stranded RNA (dsRNA) binding proteins referred to as ADARs (adenosine deaminases acting on RNA) that selectively deaminate adenosine residue(s) in precursor and mature mRNA transcripts (Nishikura, 2010). Two active members of the ADAR family, ADAR1 and ADAR2, are thought to be responsible for all mammalian A-to-I editing events and are essential for viability (Hartner et al., 2004, Higuchi et al., 2000, Wang et al., 2004). Transcription of the ADAR1 gene is complex, initiating from at least three different promoters that lead to mature mRNAs with mutually exclusive first exons (exons 1A, 1B, and 1C) via alternative splicing (Liu et al., 1997) (Fig. 1A). The promoter that initiates the ADAR1A transcript is strongly stimulated by interferon (IFN) (Der et al., 1998, George and Samuel, 1999b, Liu et al., 1997, Patterson and Samuel, 1995, Patterson et al., 1995). Translation initiates at an in-frame AUG codon at the 3′-end of exon 1A to generate a 150 kilodalton (kD) protein isoform (p150) which contains three copies of a dsRNA-binding motif (dsRBM), a motif shared among numerous dsRNA-binding proteins (Burd and Dreyfuss, 1994, Fierro-Monti and Mathews, 2000), a nuclear localization signal in the third dsRBM (Eckmann et al., 2001), and a region homologous to the catalytic domain of other known adenosine and cytidine deaminases (Fig. 1B). The amino-terminus of p150 also contains two Z-DNA binding domains, the first of which (Zα) overlaps with a leucine-rich nuclear export signal (Poulsen et al., 2001) and may be involved in the recognition of foreign nucleic acids in the cytoplasm by the innate immune system (Athanasiadis, 2012). The Z-DNA binding domains also have been proposed to tether ADAR1 to sites of transcription (Herbert and Rich, 1999, Herbert et al., 1997) or to mediate interactions between ADARs and other proteins (Poulsen et al., 2001). Transcripts initiating at constitutively expressed exons 1B or 1C use an initiation codon in exon 2 to generate a 110 kD ADAR1 protein isoform (p110) that lacks the first 248 amino acids of the p150 isoform containing the nuclear export signal and the Zα domain (George and Samuel, 1999b, Kawakubo and Samuel, 2000) (Fig. 1B). While both isoforms are capable of nuclear-cytoplasmic shuttling, they exhibit unique subcellular localizations at steady state where p110 is found predominately in nucleoli, while the majority of full-length p150 resides in the cytoplasm (Eckmann et al., 2001, Fritz et al., 2009, Patterson and Samuel, 1995, Strehblow et al., 2002). Alternative splicing within exon 7 also generates two distinct mRNA isoforms encoding ADAR1 proteins that differ by 26 amino acids in the linker region between the third double-stranded RNA binding motif and the catalytic domain (Fig. 1A) to affect site-selective editing efficiency (George et al., 2005, Liu and Samuel, 1999a, Liu et al., 1997, Liu et al., 1999a).

IFN-alpha treatment of a human glioblastoma cell line induces a robust increase in p150 expression accompanied by significant changes in the editing of 5HT_2C receptor RNAs (Yang et al., 2004). ADAR1 is also induced by a variety of inflammatory mediators including endotoxin, lipopolysaccharide, and tumor necrosis factor (Meltzer et al., 2010, Rabinovici et al., 2001, Wu et al., 2009). Oral inoculation of Salmonella in mice leads to systemic acute inflammation and expression of ADAR1 p150 RNA. However, the consequences of such treatment on ADAR1 protein levels or RNA editing patterns are not known (George et al., 2005, Shtrichman et al., 2002).

To investigate potential changes in RNA editing patterns in response to a viral CNS infection, we used reovirus serotype-3 strain Dearing (T3D) infection of neonatal mice as an experimental model system. Reoviruses are nonenveloped, icosahedral viruses with a genome consisting of 10 segments of dsRNA and are commonly used to study neurotropism and neuroinflammation (Danthi et al., 2013, Oberhaus et al., 1997, Richardson-Burns and Tyler, 2004). Reovirus is a potent inducer of type I interferon and produces a lethal meningoencephalitis in newborn animals associated with the apoptotic death of infected neurons (Danthi et al., 2008, Oberhaus et al., 1997, Richardson-Burns et al., 2002). Here we show that neonatal mice infected with reovirus T3D display large increases in p150 expression in all brain regions examined, yet the observed increase in p150 affected few editing sites. These findings suggest that steady-state editing patterns for ADAR targets are not primarily regulated by p150 expression levels.