Expression of microRNAs and their Precursors in Synaptic Fractions of Adult Mouse Forebrain
thesisposted on 07.12.2012, 00:00 by Giovanni Lugli
The expression of microRNAs and selected microRNA precursors and primary microRNA transcripts have been characterized within several synaptic fractions of adult mouse forebrain, including synaptoneurosomes, synaptosomes and isolated postsynaptic densities, 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 and primary microRNA transcripts are also detectable in synaptic fractions at levels that are at least comparable to whole tissue. Whereas mature microRNAs are predominantly associated with soluble components of the synaptic fractions, microRNA precursors and primary microRNA transcripts are predominantly associated with postsynaptic densities. I characterized a new Drosha antibody, and proved to be suitable for immunoprecipitation studies in vivo. To confirm the specificity of the Drosha antibody Drosha was silenced in N2a cells using siRNA and found that Drosha 160kDa immunoreactive band was specifically decreased. Through subcellular fractionation study, affinity purification of the postsynaptic densities and immunohistochemistry analyses of adult mouse hippocampus, I showed that Drosha and DGCR8 are localized where PSD-95 is detected. Moreover, Drosha and DGCR8 are also associated in vivo with KIF5. These findings support the proposal that microRNAs may be formed, at least in part, via processing of primary microRNA transcripts and/or microRNA precursors locally, within dendritic spines. Drosha, DGCR8 and Dicer are expressed in postsynaptic densities, whereas Dicer is enzymatically inactive until conditions that activate Calpain cause its liberation. Since Dicer is able to process primary-like microRNA transcripts and microRNA precursors, I propose that synaptic stimulation may lead to local processing of microRNA precursor-forms (pre and pri) in proximity to the synapse.