Abstract
Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation in dendrites. The in vivo roles of microRNAs in these processes are still uninvestigated, partly due to the lack of tools enabling stable in vivo delivery of microRNAs or microRNA inhibitors into neurons of the mammalian brain. Here we describe the construction and validation of a vector-based tool for stable delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming previous findings obtained with cultured neurons. Our system provides researchers with a unique tool to study the role of any candidate microRNA in vivo and can easily be adapted to microRNA loss-of-function studies. This platform should therefore greatly facilitate investigations on the role of microRNAs in synapse development, plasticity and behavior in vivo.
Highlights
MicroRNAs have emerged as important posttranscriptional regulators of gene expression
We describe the construction and validation of a vector-based tool for stable delivery of microRNAs in vivo by use of recombinant adeno-associated virus. rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layerV pyramidal neurons in vivo, thereby confirming previous findings obtained with cultured neurons
Only one strand of the miRNA duplex defines the functional miRNA, which is guided to the 3′-untranslated region (3′-UTR) of target mRNAs by the RNA-induced silencing complex (RISC)
Summary
MicroRNAs (miRNAs) have emerged as important posttranscriptional regulators of gene expression. MiRNAs bind to partially complementary regions, thereby exerting their function as regulators of gene expression (Bartel, 2004). Drosha forms a complex, the Microprocessor, with the dsRNA binding protein DGCR8 which facilitate binding of Drosha to the pri-miRNAs (Denli et al, 2004; Gregory et al, 2004; Han et al, 2004). Drosha processing requires both dsRNA elements at the base of the miRNA stem-loop structure and flanking single-stranded RNA (ssRNA) tails (Lee et al, 2003; Zeng and Cullen, 2005; Han et al, 2006)
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