Browsing by Author "Steward, Oswald"
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- Delayed Degradation and Impaired Dendritic Delivery of Intron-Lacking EGFP-Arc/Arg3.1 mRNA in EGFP-Arc Transgenic MiceSteward, Oswald; Yee, Kelly Matsudaira; Farris, Shannon; Pirbhoy, Patricia S.; Worley, Paul; Okamura, Kohji; Okuno, Hiroyuki; Bito, Haruhiko (Frontiers, 2018-01-31)Arc is a unique immediate early gene (IEG) whose expression is induced as synapses are modified during learning. Newly-synthesized ArcmRNA 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.
- Localization and local translation of Arc/Arg3.1 mRNA at synapses: some observations and paradoxesSteward, Oswald; Farris, Shannon; Pirbhoy, Patricia S.; Darnell, Jennifer; Van Driesche, Sarah J. (Frontiers, 2015-01-12)Arc is a unique immediate early gene whose expression is induced as synapses are being modified during learning. The uniqueness comes from the fact that newly synthesized Arc mRNA is rapidly transported throughout dendrites where it localizes near synapses that were recently activated. Here, we summarize aspects of Arc mRNA translation in dendrites in vivo, focusing especially on features of its expression that are paradoxical or that do not fit in with current models of how Arc protein operates. Findings from in vivo studies that donor quite fit include: (1) Following induction of LTP in vivo, Arc mRNA and protein localize near active synapses, but are also distributed throughout dendrites. In contrast, Arc mRNA localizes selectively near active synapses when stimulation is continued as Arc mRNA is transported into dendrites; (2) Strong induction of Arc expression as a result of a seizure does not lead to a rundown of synaptic efficacy in vivo as would be predicted by the hypothesis that high levels of Arc cause glutamate receptor endocytosis and LTD. (3) Arc protein is synthesized in the perinuclear cytoplasm rapidly after transcriptional activation, indicating that at least a pool of Arc mRNA is not translationally repressed to allow for dendritic delivery; (4) Increases in Arc mRNA in dendrites are not paralleled by increases in levels of exon junction complex (EJC) proteins. These results of studies of mRNA trafficking in neurons in vivo provide a new perspective on the possible roles of Arc in activity-dependent synaptic modifications.