Delayed Degradation and Impaired Dendritic Delivery of Intron-Lacking EGFP-Arc/Arg3.1 mRNA in EGFP-Arc Transgenic Mice

Abstract

Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized Arc mRNA is rapidly transported throughout dendrites and localizes near recently activated synapses. Arc mRNA levels are regulated by rapid degradation, which is accelerated by synaptic activity in a translation-dependent process. One possible mechanism is nonsense-mediated mRNA decay (NMD), which depends on the presence of a splice junction in the 3′UTR. Here, we test this hypothesis using transgenic mice that express EGFP-Arc. Because the transgene was constructed from Arc cDNA, it lacks intron structures in the 3′UTR that are present in the endogenous Arc gene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, so EGFP-Arc mRNA should not undergo NMD. Assessment of Arc mRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation of EGFP-Arc mRNA. EGFP-Arc mRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions but EGFP-Arc mRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction of Arc by seizures, there were increases in endogenous Arc mRNA levels consistent with translation-dependent Arc mRNA decay but this was not seen with EGFP-Arc mRNA. Taken together, our results indicate: (1) Arc mRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesized Arc mRNA after induction may depend in part on prior splicing of the 3′UTR.

Arc 3′ UTR Splicing Leads to Dual and Antagonistic Effects in Fine-Tuning Arc Expression Upon BDNF Signaling

“…Confirmation that Arc 3′ UTR splicing downregulates Arc mRNA abundance upon neuronal activation was also recently provided in vivo ( Steward et al, 2018 ), adopting EGFP-Arc transgenic mice with an intron-less 3′ UTR region. Additionally, Steward and colleagues find that 3′ UTR splicing is required for efficient dendritic delivery of Arc mRNA.…”

“…In the absence of inducing stimuli Arc mRNA and protein are almost undetectable in vivo and in vitro ( Giorgi et al, 2007 ; Steward et al, 2014 , 2018 ). However, its transcription and translation are strongly enhanced by several plasticity inducing stimuli including exogenous addition of BDNF to in vitro neuronal cultures ( Bramham et al, 2010 ; El-Sayed et al, 2011 ; Leal et al, 2014 ).…”

“…The 3′ UTR region of messenger RNAs plays a central role in coordinating their post-transcriptional regulation, recruiting RNA binding factors that affect their localization, stability, and translation ( Giorgi and Moore, 2007 ; Gebauer et al, 2012 ; Matoulkova et al, 2012 ). Arc mRNA 3′ UTR is particularly interesting as it harbors two conserved introns, which regulate its abundance targeting it for destruction via the NMD ( Giorgi et al, 2007 ; Steward et al, 2018 ). This feature is extremely unusual among eukaryotic genes and its implications in Arc gene expression have not been fully elucidated.…”

“…The subcellular localization of this translational modulation is another aspect that necessitates further investigation, particularly in light of recent evidences that link Arc 3′ UTR splicing with its proper dendritic localization ( Steward et al, 2018 ).…”

Intercellular Arc signaling regulates vasodilation

“…As a control for the specificity of our in situ hybridization probes, we repeated our experiments with a negative control RNA probe for Arc and found little to no detectable signal in either the DRG or spinal dorsal horn ( Figure S2A-C). Reporter mice have been generated that express a translational fusion between Arc and eGFP (Steward et al, 2017). Arc-GFP is present in DRG neurons and in sciatic nerve fibers in these animals (Unpublished observation).…”