MicroRNAs are known to be involved in the development of the nervous system and to act by suppressing gene expression. The authors used RNA interference (RNAi) to downregulate Dicer-1 (an enzyme that is essential for microRNA synthesis) in postsynaptic muscle cells in the flies. Using reverse transcription PCR and fluorescence in situ hybridization, two methods that allow the quantification of RNA transcripts, the authors found that there were considerably higher levels of expression of the GluR subunit isoform genes
GluRIIA
and
GluRIIB
in the NMJ of RNAi flies, indicating that these transcripts are normally repressed by microRNAs. Convergently, immunocytochemistry showed increased levels of GluRIIA and GluRIIB proteins, most of which, however, were not localized to the postsynaptic membrane. Electrophysiological investigation confirmed that the changes in protein abundance did not significantly affect total synaptic strength.
Next, the authors used finely tuned bioinformatic models to predict microRNA binding sites and identified binding sites for miR-284 in both GluRIIA and GluRIIB transcripts. In miR-284 loss-of-function mutants they found that GluRIIB mRNA, which has two predicted binding sites, was de-repressed more than GluRIIA mRNA, which has one. Furthermore, transgenic expression of miR-284 in these mutant flies restored GluRIIA and GluRIIB levels to near control levels, thereby confirming that this microRNA directly affected protein abundance, in proportion to the number of miR-284 binding sites on the transcript subtypes.
This is a preview of subscription content, access via your institution
