Small RNAs: Their applications and biological functions in neural development.
Jenn‐Yah Yu · Deep Blue (University of Michigan) · 2006
Recently, research on different organisms has revealed a new layer of gene regulation by small RNAs, which have sequence-specific inhibitory effects on gene expression. Long double-stranded RNAs are processed into small interfering RNAs (shRNAs), which are incorporated into the RNA-induced silencing complex and mediate target mRNA cleavage. Synthetic shRNAs also inhibit gene expression and have become effective tools for studying gene function. As an alternative to synthetic shRNAs, we designed DNA vectors expressing short hairpin RNAs (shRNAs) in cells. Designs for shRNAs were evaluated using in vitro transcription from oligonucleotide templates. An shRNA expression vector with a U6 RNA polymerase III promoter expressed shRNAs with defined ends and inhibited gene expression efficiently. We further examined the effects of length and loop sequence on inhibition by shRNAs to optimize our designs. Cotransfected shRNA vectors do not interfere with each other, and two genes can be inhibited simultaneously. We also tested shRNA vectors in dissociated cortical primary neurons and organotypic brain slices. Our results show that the use of shRNA vectors provides a versatile approach for analyzing gene function during neural development. MicroRNAs (miRNAs) are endogenous non-coding small RNAs with diverse regulatory roles. Most miRNAs interact with 3' untranslated region of the target genes through sequence complementarity and regulate the stability or translational efficiency of target mRNAs. We found that miRNA miR-124a affects neurite outgrowth during neuronal differentiation. Expression of miR-124a in differentiating P19 cells promoted neurite outgrowth, while blocking of miR-124a function delayed neurite outgrowth. In uncommitted P19 cells, miR-124a led to disruption of actin filaments and stabilization of microtubules, which resemble the cytoskeleton remodeling during neuronal morphogenesis. Rho GTPases, including Rac, Rho, and Cdc42, affect neuronal development through regulation of microfilaments and microtubules. miR-124a decreased protein levels of Cdc42 and affected subcellular localization of Rac1. Furthermore, inhibition of Rac1 activities in differentiating P19 cells promoted neurite outgrowth; and activation of either Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124a. These results suggest that Cdc42 and Rac1 may function downstream of miR-124a in promoting neurite outgrowth. Altered neurite outgrowth was also observed in mouse primary cortical neurons when the expression level of miR-124a was increased, or when miR-124a function was blocked. These results indicate that miRNAs can contribute to the regulation of neurite outgrowth in neuronal differentiation.