GluA2 mRNA distribution and regulation by miR-124 in hippocampal neurons.
AMPA-type glutamate receptors mediate fast, excitatory neurotransmission in the brain, and their concentrations at synapses are important determinants of synaptic strength. We investigated the post-transcriptional regulation of GluA2, the calcium-impermeable AMPA receptor subunit, by examining the subcellular distribution of its mRNA and evaluating its translational regulation by microRNA in cultured mouse hippocampal neurons. Using computational approaches, we identified a conserved microRNA-124 (miR-124) binding site in the 3'UTR of GluA2 and demonstrated that miR-124 regulated the translation of GluA2 mRNA reporters in a sequence-specific manner in luciferase assays. While we hypothesized that this regulation might occur in dendrites, our biochemical and fluorescent in situ hybridization (FISH) data indicate that GluA2 mRNA does not localize to dendrites or synapses of mouse hippocampal neurons. In contrast, we detected significant concentrations of miR-124 in dendrites. Overexpression of miR-124 in dissociated neurons results in a 30% knockdown of GluA2 protein, as measured by immunoblot and quantitative immunocytochemistry, without producing any changes in GluA2 mRNA concentrations. While total GluA2 concentrations are reduced, we did not detect any changes in the concentration of synaptic GluA2. We conclude from these results that miR-124 interacts with GluA2 mRNA in the cell body to downregulate translation. Our data support a model in which GluA2 is translated in the cell body and subsequently transported to neuronal dendrites and synapses, and suggest that synaptic GluA2 concentrations are modified primarily by regulated protein trafficking rather than by regulated local translation.
Interdepartmental Program for Neuroscience, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA.
Victoria M
VM
Ho
Interdepartmental Program for Neuroscience, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA.
Liane O
LO
Dallalzadeh
Department of Biology, University of Crete, Heraklion, Crete, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, Greece.
Nestoras
N
Karathanasis
Interdepartmental Program for Molecular, Cellular and Integrative Physiology, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA.
Mehmet F
MF
Keles
Department of Medicine Statistics Core, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Sitaram
S
Vangala
Department of Medicine Statistics Core, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Tristan
T
Grogan
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, Greece.
Panayiota
P
Poirazi
Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA; Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA; Integrated Center for Learning and Memory, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA. Electronic address: kcmartin@mednet.ucla.edu.
Kelsey C
KC
Martin
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D003712:Dendrites / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D006624:Hippocampus / Q000166:cytology*; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010852:Picrotoxin / Q000494:pharmacology; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D018091:Receptors, AMPA / Q000235:genetics* / Q000378:metabolism; D054387:Receptors, CXCR / Q000235:genetics / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism
D000697:Central Nervous System Stimulants; D035683:MicroRNAs; D009419:Nerve Tissue Proteins; D010852:Picrotoxin; D012333:RNA, Messenger; D018091:Receptors, AMPA; D054387:Receptors, CXCR; C540079:Mirn124 microRNA, mouse; C104722:glutamate receptor ionotropic, AMPA 2
AMPAR; Dendritic mRNA localization; Local translation; MicroRNA; Post-transcriptional regulation; miR-124