Activity-Dependent A-to-I RNA Editing in Rat Cortical Neurons
Sanjana, Neville E., Levanon, Erez Y., Hueske, Emily A., Ambrose, Jessica M., Li, Jin Billy, Genetics
ABSTRACT Changes in neural activity influence synaptic plasticity/scaling, gene expression, and epigenetic modifications. We present the first evidence that short-term and persistent changes in neural activity can alter adenosine-to-inosine (A-to-I) RNA editing, a post-transcriptional site-specific modification found in several neuron-specific transcripts. In rat cortical neuron cultures, activity-dependent changes in A-to-I RNA editing in coding exons are present after 6 hr of high potassium depolarization but not after 1 hr and require calcium entry into neurons. When treatments are extended from hours to days, we observe a negative feedback phenomenon: Chronic depolarization increases editing at many sites and chronic silencing decreases editing. We present several different modulations of neural activity that change the expression of different mRNA isoforms through editing.
CHANGES in neural activity can modulate synaptic strength at the level of individual neurons (Burrone et al. 2002) and whole networks (Turrigiano et al. 1998), influence developmental and differentiation decisions (Borodinsky et al. 2004), and bias neurons toward inclusion in novel memory formation (Zhou et al. 2009). At the mRNA level, immediate early genes (IEGs) are transcribed rapidly in response to increased neural activity and have been described as a "genomic action potential" (Clayton 2000). The promoter regions of these and other genes involved in neuronal plasticity can undergo activity-dependent alterations in their chromatin structure, such as cytosine demethylation and histone acetylation (Tsankova et al. 2004; Ma et al. 2009). In addition to the major, genome-wide epigenetic changes known to occur during early development, recent studies have found activity-driven epigenetic modifications in post-mitotic neurons during fear memory recall, exposure to drugs of abuse, and emotional stress (Renthal et al. 2007). We sought to understand if neural activity influences a post-transcriptional genetic modification, adenosine-toinosine (A-to-I) RNA editing.
A-to-I RNA editing is an enzymatically catalyzed, sitespecific nucleotide change (deamination) in pre-mRNA that changes adenosine into inosine, which reads as guanosine during translation (Bass 2002; Nishikura 2010). The bestknown example is the developmentally regulated editing of the glutamate receptor subunit Gria2, where A-to-I editing at a single site controls whether GRIA2-containing AMPA receptors are permeable to Ca2+ ions (Sommer et al. 1991). In neurons, many transcripts undergo editing: Previous large-scale genomic screens in Drosophila and human cells found that synapse-related transcripts are enriched in RNA-editing sites (Paul and Bass 1998; Hoopengardner et al. 2003; Li et al. 2009). More generally, inosine is greatly enriched in brain tissue compared to other tissues (Paul and Bass 1998) and the mammalian adenosine deaminases acting on RNA (ADARs) tend to be preferentially or exclusively expressed in the brain (Melcher et al. 1996). Knockout of ADARs in mice, Drosophila, and Caenorhabditis elegans leads to aberrant neurological and behavioral phenotypes (Brusa et al. 1995; Tonkin et al. 2002; Savva et al. 2012). This strongly supports that RNA editing influences neural function, but it is not well understood whether changes in neural activity can affect RNA editing to exert control over transcript diversity and protein function. Previously, it has been shown that editing at two sites in serotonin 2C (Htr2c) pre-mRNA is regulated in a serotonin-dependent manner without any change in transcript expression levels (Gurevich et al. 2002); early life stress and the common antidepressant fluoxetine can also alter Htr2c editing (Englander et al. 2005; Bhansali et al. 2007). These studies suggest a possible role for neural activity in inducing changes in RNA editing, but this connection has not been systematically explored on a genome-wide scale with newer sequencing techniques. …