Regulation and function of microRNA during neural development and stem cell specification

In the present study the role of microRNAs (miRNAs) in the control of developmental timing in the mammalian nervous system and in the specification of neural cell fate was investigated. miRNAs are a recently discovered class of small, 21-22 nt, regulatory RNA molecules. They inhibit translation of target mRNAs by binding to sites of imperfect anti-sense complementarity in 3? untranslated regions (UTRs). Many miRNAs are evolutionarily conserved, which has allowed their identification in various species. In the model organisms C. elegans and D. melanogaster, miRNAs regulate genes involved in fundamental developmental processes including cell proliferation, apoptosis, and the timing of cell fate decisions in the CNS (e.g. let-7 and lin-4 for C. elegans and Bantam and mir-14 for D. melanogaster). Hundreds of miRNA genes are expressed in humans and mice, and a substantial fraction of these genes has been identified in neural cells. Although the biological functions of most miRNAs are unknown, miRNAs are predicted to regulate about 30% of the human genes. Disruption of miRNA biogenesis is definitely associated with severe disturbances in neural development in model organisms and most likely with human clinical syndromes (Fragile X Mental Retardation Syndrome, Spinal Motor Atrophy, DiGeorge Syndrome). This fact, together with the well established role of miRNA genes in C. elegans and D. melanogaster development, points to the relevance of this newly emerging field for the understanding of developmental disorders. In this work the regulation of a set of highly expressed neural miRNAs, and in particular the let-7 family during mouse brain development and neural differentiation of embryonic stem (ES) cells has been studied. Significant differences were observed in the onset and magnitude of induction for individual miRNAs. miRNAs were strongly induced during neural differentiation of ES cells, suggesting the validity of the stem cell model for studying miRNA regulation in neural development. In undifferentiated ES and embryonal carcinoma (EC) cells, both the let-7 primary transcript and precursor were detected in the absence of mature miRNA accumulation, suggesting an important post-transcriptional component in the regulation of let-7 expression. An in vitro assay for precursor processing revealed developmental regulation of let-7 as well as mir-128 and mir-30 maturation. Precursor processing activity increased during neural differentiation of ES and EC cells and was greater in primary neurons compared to astrocytes. Neuron-specific binding activity of pre-miRNAs was shown by antibody challenge to contain the Fragile X Mental Retardation Protein (FMRP). As further evidence for developmental regulation of the miRNA processing pathway, it was shown that Argonaute proteins and FMRP failed to localize to cytoplasmic foci identified as processing bodies (P-bodies) in self-renewing ES or EC cells. Comparing expression in cultures of embryonic neurons and astrocytes, marked lineage specificity was found for many of the miRNAs studied. Two of the most highly expressed miRNAs in adult brain (mir-124, mir-128) were preferentially expressed in neurons. In contrast, mir-23, a miRNA previously implicated in neural specification, was restricted to astrocytes. Lineage specificity was further explored using reporter constructs for three miRNAs of particular interest (let-7, mir-125 and mir-128). miRNA-mediated suppression of these reporters was observed after their transfection into neurons but not astrocytes. Furthermore, reporter constructs containing let-7 or mir-125 target sites were downregulated in EC-derived neurons, reflecting the upregulation of miRNAs during neuronal development. In addition, mRNA target degradation was observed in response to let-7 and mir-125, opening new questions regarding the mechanism of miRNA-mediated mRNA silencing. Disrupting the interaction of let-7 and mir-125 with their target genes during neural differentiation led to an increase in astrocyte marker expression (GFAP and A2B5), implicating let 7 and mir-125 in neuronal lineage commitment. Finally, a functional let-7/mir-125 response element in the 3? UTR of a mouse lin-41 homolog was identified, revealing a conserved let-7/target gene interaction that is active during early neural differentiation.