Jcb_200902062 685..698

Most synaptic transmission in mammalian brains occurs via glutamate receptors (GluRs). Synthesis and localization of GluRs is essential for brain development, and GluR changes underlie learning, memory, and many different neuropathologies. Several lines of evidence suggest that GluR synthesis may be regulated by microRNAs, which suppress gene expression in a variety of organisms and tissue types (Lee et al., 2007; Baek et al., 2008; Selbach et al., 2008). First, microRNAs are particularly abundant in brains, and have recently been shown to play important roles in several aspects of nervous system development (Cao et al., 2006; Bicker and Schratt, 2008; Fiore et al., 2008). Second, some postsynaptic proteins, possibly including GluR subunits, seem to be locally translated near postsynaptic densities (Sigrist et al., 2000; Job and Eberwine, 2001; Miller et al., 2002; Sutton et al., 2004; Sutton and Schuman, 2005; Martin and Zukin, 2006; Pfeiffer and Huber, 2006; Schuman et al., 2006; Bramham, 2008), whereas the microRNA synthesis protein dicer and many microRNAs themselves may be enriched at synapses (Lugli et al., 2005, 2008). Finally, acetylcholine receptor abundance in Caenorhabditis elegans neuromuscular junctions (NMJs) has recently been shown to be regulated by microRNAs (Simon et al., 2008). Here, we test whether GluR synthesis in Drosophila melanogaster is regulated by microRNAs. Drosophila NMJs are glutamatergic synapses that are individually identifiable at the cellular level and experimentally accessible throughout development in vivo (Keshishian et al., 1996; Featherstone and Broadie, 2004; Ruiz-Canada and Budnik, 2006). Drosophila NMJs contain two biophysically, pharmacologically, and spatially distinct subtypes of ionotropic GluRs (DiAntonio et al., 1999; Marrus et al., 2004; Chen et al., 2005; Featherstone et al., 2005; Qin et al., 2005), called “A type” and “B type.” A-type receptors contain the subunit GluRIIA but not GluRIIB, plus the subunits GluRIIC (also known as GluRIII), GluRIID, and GluRIIE. B-type receptors contain GluRIIB but not GluRIIA, plus GluRIIC, GluRIID, and GluRIIE. Our results show that microRNAs strongly regulate the abundance of GluRIIA and GluRIIB GluR subunit mRNAs, and that this has dramatic effects on relative subunit protein abundance. In the absence of microRNA-mediated subunit repression, The efficacy of synaptic transmission depends, to a large extent, on postsynaptic receptor abundance. The molecular mechanisms controlling receptor abundance are poorly understood. We tested whether abundance of postsynaptic glutamate receptors (GluRs) in Drosophila neuromuscular junctions is controlled by microRNAs, and provide evidence that it is. We show here that postsynaptic knockdown of dicer-1, the endoribonuclease necessary for microRNA synthesis, leads to large increases in postsynaptic GluR subunit messenger RNA and protein. Specifically, we measured increases in GluRIIA and GluRIIB but not GluRIIC. Further, knockout of MiR-284, a microRNA predicted to bind to GluRIIA and GluRIIB but not GluRIIC, increases expression of GluRIIA and GluRIIB but not GluRIIC proportional to the number of predicted binding sites in each transcript. Most of the derepressed GluR protein, however, does not appear to be incorporated into functional receptors, and only minor changes in synaptic strength are observed, which suggests that microRNAs primarily regulate Drosophila receptor subunit composition rather than overall receptor abundance or synaptic strength. Regulation of glutamate receptor subunit availability by microRNAs