Abstract
We have characterized the expression of microRNAs and selected microRNA precursors within several synaptic fractions of adult mouse forebrain, including synaptoneurosomes, synaptosomes and isolated post-synaptic densities (PSDs), using methods of microRNA microarray, real time qRT-PCR, Northern blotting and immunopurification using anti-PSD95 antibody. The majority of brain microRNAs (especially microRNAs known to be expressed in pyramidal neurons) are detectably expressed in synaptic fractions, and a subset of microRNAs is significantly enriched in synaptic fractions relative to total forebrain homogenate. MicroRNA precursors are also detectable in synaptic fractions at levels that are comparable to whole tissue. Whereas mature microRNAs are predominantly associated with soluble components of the synaptic fractions, microRNA precursors are predominantly associated with PSDs. For seven microRNAs examined, there was a significant correlation between the relative synaptic enrichment of the precursor and the relative synaptic enrichment of the corresponding mature microRNA. These findings support the proposal that microRNAs are formed, at least in part, via processing of microRNA precursors locally within dendritic spines. Dicer is expressed in PSDs but is enzymatically inactive until conditions that activate calpain cause its liberation; thus, we propose that synaptic stimulation may lead to local processing of microRNA precursors in proximity to the synapse.
Highlights
The rate-limiting enzyme in formation of small RNAs, is expressed in the somatodendritic domain of projection neurons, including the dendritic spines, and is enriched at postsynaptic densities (PSDs) (Lugli, 2005). Using immunocytochemistry at both LM (Figure 2) and EM levels (Figure 3), which was performed by Martone M.E. and Jones Y., we showed that Dicer and eIF2c (Argonaute homologue) were expressed mainly in the somatodendritic compartment of neurons, and were detectable as well within dendritic spines
Synaptoneurosomes are small vesicular structures that are prepared by filtration of total forebrain homogenate through mesh of progressively decreasing size, followed by lowspeed centrifugation and higher-speed centrifugation
Synaptoneurosomal preparations were characterized by Western blotting of PSD95 and by real-time RT-PCR measurements of BC1 and CAMK2a to verify that these components were enriched relative to total forebrain homogenate
Summary
Discovery of microRNAs In 1993, Lee (1993), discovered that lin-4 RNA, a gene known to control the timing of Caenorhabditis elegans (C. elegans) larval development, does not code for a protein but instead produces a pair of small RNAs and negatively regulates the translation of LIN-14 protein This discovery can be considered the first evidence of the existence of what we know as microRNAs. This discovery can be considered the first evidence of the existence of what we know as microRNAs They suggested that because the lin-4 sequence had complementarity to the lin-14 3’UTR region, a possible mechanism of action to be an antisense RNA-RNA interaction.
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