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

Oswald Steward, Kelly Matsudaira Yee, Shannon Farris, Patricia S. Pirbhoy, Paul Worley, Kohji Okamura, Hiroyuki Okuno, Haruhiko Bito

Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesizedArcmRNA is rapidly transported throughout dendrites and localizes near recently activated synapses.ArcmRNA 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 expressEGFP-Arc. Because the transgene was constructed fromArccDNA, it lacks intron structures in the 3'UTR that are present in the endogenousArcgene. NMD depends on the presence of proteins of the exon junction complex (EJC) downstream of a stop codon, soEGFP-Arc mRNAshould not undergo NMD. Assessment ofArcmRNA rundown in the presence of the transcription inhibitor actinomycin-D confirmed delayed degradation ofEGFP-ArcmRNA.EGFP-ArcmRNA and protein are expressed at much higher levels in transgenic mice under basal and activated conditions butEGFP-ArcmRNA does not enter dendrites efficiently. In a physiological assay in which cycloheximide (CHX) was infused after induction ofArcby seizures, there were increases in endogenousArcmRNA levels consistent with translation-dependentArcmRNA decay but this was not seen withEGFP-ArcmRNA. Taken together, our results indicate: (1)ArcmRNA degradation occurs via a mechanism with characteristics of NMD; (2) rapid dendritic delivery of newly synthesizedArcmRNA after induction may depend in part on prior splicing of the 3'UTR.