The neuronal RNA-binding protein HuD plays a critical role in the post-transcriptional regulation of short-lived mRNAs during the initial establishment and remodelling of neural connections. We have generated transgenic mice overexpressing this protein (HuD-Tg) in adult DGCs (dentate granule cells) and shown that their mossy fibres contain high levels of GAP-43 (growth-associated protein 43) and exhibit distinct morphological and electrophysiological properties. To investigate the basis for these changes and identify other molecular targets of HuD, DGCs from HuD-Tg and control mice were collected by LCM (laser capture microscopy) and RNAs analysed using DNA microarrays. Results show that 216 known mRNAs transcripts and 63 ESTs (expressed sequence tags) are significantly up-regulated in DGCs from these transgenic mice. Analyses of the 3′-UTRs (3′-untranslated regions) of these transcripts revealed an increased number of HuD-binding sites and the presence of several known instability-conferring sequences. Among these, the mRNA for TTR (transthyretin) shows the highest level of up-regulation, as confirmed by qRT–PCR (quantitative reverse transcription–PCR) and ISH (in situ hybridization). GO (gene ontology) analyses of up-regulated transcripts revealed a large over-representation of genes associated with neural development and axogenesis. In correlation with these gene expression changes, we found an increased length of the infrapyramidal mossy fibre bundle in HuD-Tg mice. These results support the notion that HuD stabilizes a number of developmentally regulated mRNAs in DGCs, resulting in increased axonal elongation.
Keywords: axonal outgrowth, dentate granule cell, gene profiling, HuD, post-transcriptional mechanisms, RNA-binding protein
Abbreviations: αCaMKII, α-Ca2+/calmodulin-dependent protein kinase II, ARE, AU-rich element, ARED, ARE database, CP, choroid plexus, Dcx, doublecortin, DGC, dentate granule cell, DIG, digoxigenin, EST, expressed sequence tag, FISH, fluorescence in situ hybridization, GAP-43, growth-associated protein 43, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GO, gene ontology, GRE, GU-rich element, IPA, ingenuity pathway analysis, IPB, infrapyramidal bundle, ISH, in situ hybridization, KO, knockout, LCM, laser capture microscopy, Mtap1b, microtubule-associated protein 1b, NIH, National Institutes of Health, PFA, paraformaldehyde, PPF, paired-pulse facilitation, qRT–PCR, quantitative reverse transcription–PCR, RNA-IP, RNA-immunoprecipitation, SMA, spinal muscular atrophy, SPB, suprapyramidal bundle, T3, 3,3′,5-tri-iodothyronine, T4, thyroxine, TTR, transthyretin, 3′-UTR, 3′-untranslated region, WT, wild-type