Reciprocal regulation of A-to-I RNA editing and the vertebrate nervous system

The fine control of molecules mediating communication in the nervous system is key to adjusting neuronal signaling during development and in maintaining the stability of established networks in the face of altered sensory input. To prevent the culmination of pathological recurrent network excitation or debilitating periods of quiescence, adaptive alterations occur in the signaling molecules and ion channels that control membrane excitability and synaptic transmission. However, rather than encoding (and thus "hardwiring") modified gene copies, the nervous systems of metazoa have opted for expanding on post-transcriptional pre-mRNA splicing by altering key encoded amino acids using a conserved mechanism of A-to-I RNA editing: the enzymatic deamination of adenosine to inosine. Inosine exhibits similar base-pairing properties to guanosine with respect to tRNA codon recognition, replication by polymerases, and RNA secondary structure (i.e.,: forming-capacity). In addition to recoding within the open reading frame, adenosine deamination also occurs with high frequency throughout the non-coding transcriptome, where it affects multiple aspects of RNA metabolism and gene expression. Here, we describe the recoding function of key RNA editing targets in the mammalian central nervous system and their potential to be regulated. We will then discuss how interactions of A-to-I editing with gene expression and alternative splicing could play a wider role in regulating the neuronal transcriptome. Finally, we will highlight the increasing complexity of this multifaceted control hub by summarizing new findings from high-throughput studies. © 2013 Penn, Balik and Greger

AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions.

AMPA receptor (AMPAR) abundance and positioning at excitatory synapses regulates the strength of transmission. Changes in AMPAR localisation can enact synaptic plasticity, allowing long-term information storage, and is therefore tightly controlled. Multiple mechanisms regulating AMPAR synaptic anchoring have been described, but with limited coherence or comparison between reports, our understanding of this process is unclear. Here, combining synaptic recordings from mouse hippocampal slices and super-resolution imaging in dissociated cultures, we compare the contributions of three AMPAR interaction domains controlling transmission at hippocampal CA1 synapses. We show that the AMPAR C-termini play only a modulatory role, whereas the extracellular N-terminal domain (NTD) and PDZ interactions of the auxiliary subunit TARP γ8 are both crucial, and each is sufficient to maintain transmission. Our data support a model in which γ8 accumulates AMPARs at the postsynaptic density, where the NTD further tunes their positioning. This interplay between cytosolic (TARP γ8) and synaptic cleft (NTD) interactions provides versatility to regulate synaptic transmission and plasticity

Activity-mediated AMPA receptor remodeling, driven by alternative splicing in the ligand-binding domain

The AMPA-type glutamate receptor (AMPAR) subunit composition shapes synaptic transmission and varies throughout development and in response to different input patterns. Here, we show that chronic activity deprivation gives rise to synaptic AMPAR responses with enhanced fidelity. Extrasynaptic AMPARs exhibited changes in kinetics and pharmacology associated with splicing of the alternative flip/flop exons. AMPAR mRNA indeed exhibited reprogramming of the flip/flop exons for GluA1 and GluA2 subunits in response to activity, selectively in the CA1 subfield. However, the functional changes did not directly correlate with the mRNA expression profiles but result from altered assembly of GluA1/GluA2 subunit splice variants, uncovering an additional regulatory role for flip/flop splicing in excitatory signaling. Our results suggest that activity-dependent AMPAR remodeling underlies changes in short-term synaptic plasticity and provides a mechanism for neuronal homeostasis

Activity-regulated RNA editing in select neuronal subfields in hippocampus

RNA editing by adensosine deaminases is a widespread mechanism to alter genetic information in metazoa. In addition to modifications in non-coding regions, editing contributes to diversification of protein function, in analogy to alternative splicing. However, although splicing programs respond to external signals, facilitating fine tuning and homeostasis of cellular functions, a similar regulation has not been described for RNA editing. Here, we show that the AMPA receptor R/G editing site is dynamically regulated in the hippocampus in response to activity. These changes are bi-directional, reversible and correlate with levels of the editase Adar2. This regulation is observed in the CA1 hippocampal subfield but not in CA3 and is thus subfield/celltype-specific. Moreover, alternative splicing of the flip/flop cassette downstream of the R/G site is closely linked to the editing state, which is regulated by Ca(2+). Our data show that A-to-I RNA editing has the capacity to tune protein function in response to external stimuli

Steric antisense inhibition of AMPA receptor Q/R editing reveals tight coupling to intronic editing sites and splicing

Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional mechanism, evolved to diversify the transcriptome in metazoa. In addition to wide-spread editing in non-coding regions protein recoding by RNA editing allows for fine tuning of protein function. Functional consequences are only known for some editing sites and the combinatorial effect between multiple sites (functional epistasis) is currently unclear. Similarly, the interplay between RNA editing and splicing, which impacts on post-transcriptional gene regulation, has not been resolved. Here, we describe a versatile antisense approach, which will aid resolving these open questions. We have developed and characterized morpholino oligos targeting the most efficiently edited site--the AMPA receptor GluA2 Q/R site. We show that inhibition of editing closely correlates with intronic editing efficiency, which is linked to splicing efficiency. In addition to providing a versatile tool our data underscore the unique efficiency of a physiologically pivotal editing site

AMPA receptor biogenesis and trafficking.

AMPA-type glutamate receptors mediate the majority of fast excitatory transmission in the central nervous system. The trafficking of AMPA receptors to and from synapses alters synaptic strength and has been recognized as a central mechanism underlying various forms of synaptic plasticity. Both secretory and endocytic trafficking events seem to be driven by the subunit composition of AMPA receptor tetramers. Moreover, recent work suggests that synapses employ different tetramer combinations in response to altered synaptic input, suggesting the existence of signalling pathways that mediate remodelling of AMPA receptors. These latest developments and recent progress in elucidating the mechanisms that underlie channel assembly and trafficking are the subject of this review.We acknowledge the support of the Royal Society and the MRC (IHG), and NIH grant MH070417 (JAE).Peer reviewe

AMPA receptor assembly: atomic determinants and built-in modulators

Glutamate-gated ion channels (iGluRs) predominantly operate as heterotetramers to mediate excitatory neurotransmission at glutamatergic synapses. The subunit composition of the receptors determines their targeting to synaptic sites and signalling properties and is therefore a fundamental parameter for neuronal computations. iGluRs assemble as obligatory or preferential heteromers; the mechanisms underlying this selective assembly are only starting to emerge. Here we review recent work in the field and provide an in-depth update on atomic determinants in the assembly domains, which have been facilitated by recent advances in iGluR structural biology. We also discuss the role of alternative RNA processing in the ligand-binding domain, which modulates a central subunit interface and has the capacity to modulate receptor formation in response to external cues. Finally, we review the emerging physiological significance of signalling via distinct iGluR heterotetramers and provide examples of how recruitment of functionally diverse receptors modulates excitatory neurotransmission under physiological and pathological conditions. © 2012 Springer-Verlag/WIen

Molecular determinants of AMPA receptor subunit assembly

AMPA-type (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamate receptors (AMPARs) mediate post-synaptic depolarization and fast excitatory transmission in the central nervous system. AMPARs are tetrameric ion channels that assemble in the endoplasmic reticulum (ER) in a poorly understood process. The subunit composition determines channel conductance properties and gating kinetics, and also regulates vesicular traffic to and from synaptic sites, and is thus critical for synaptic function and plasticity. The distribution of functionally different AMPARs varies within and between neuronal circuits, and even within individual neurons. In addition, synapses employ channels with specific subunit stoichiometries, depending on the type of input and the frequency of stimulation. Taken together, it appears that assembly is not simply a stochastic process. Recently, progress has been made in understanding the molecular mechanisms underlying subunit assembly and receptor biogenesis in the ER. These processes ultimately determine the size and shape of the postsynaptic response, and are the subject of this review