AB5971 Sigma-AldrichAnti-HuD Antibody

Little is known about how a neuron undergoes site-specific changes in intrinsic excitability during neuronal activity. We provide evidence for a novel mechanism for mTORC1 kinase-dependent translational regulation of the voltage-gated potassium channel Kv1.1 messenger RNA (mRNA). We identified a microRNA, miR-129, that repressed Kv1.1 mRNA translation when mTORC1 was active. When mTORC1 was inactive, we found that the RNA-binding protein, HuD, bound to Kv1.1 mRNA and promoted its translation. Unexpectedly, inhibition of mTORC1 activity did not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA. However, reduced mTORC1 signaling caused the degradation of high affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Hence, mTORC1 activity regulation of mRNA stability and high affinity HuD-target mRNA degradation mediates the bidirectional expression of dendritic Kv1.1 ion channels.

The 3'-untranslated regions (3'-UTRs) of growth-associated protein 43 (GAP-43), which is crucial for neural development and axonal regeneration, are highly conserved among vertebrates. Previous studies in mammals have identified one U-rich cis element within GAP-43 3'-UTR and several trans factors that regulate its mRNA stability. However, much less is known in lower vertebrates. The Xenopus GAP-43 3'-UTR, despite its high similarity with those in higher vertebrates, contains unique CU-rich sequences, suggesting the existence of novel cis elements and trans factors. In current study, we isolated four proteins bound to GAP-43 3'-UTR from juvenile frog brain using affinity purification. Mass spectrometry identified Hu antigen D (HuD) and poly(C) binding protein 2 (αCP2) as the proteins forming 48- and 44-kDa ribonucleoprotein complexes, respectively. We validated the association between αCP2 and GAP-43 3'-UTR in vivo. After confirming the post-transcriptional effects of αCP2 on GAP-43 expression, we demonstrated that αCP2 directly inhibited the translation of GAP-43 gene, without affecting its mRNA stability. αCP2 overexpression led to decreased level of GAP-43 protein and significantly inhibited axonal outgrowth in primarily cultured neurons. Our study therefore provided insights on novel functions of αCP2 in vertebrate nervous system during development and new mechanisms of post-transcriptional regulation for GAP-43 gene.