Previous studies have identified three brain proteins (40, 65 and 95 kDa, respectively) that specifically bind to the 3-untranslated region of GAP-43 mRNA. In this study, using a specific monoclonal antibody, we now show that the 40-kDa proteins are members of the Elavlike protein family. This family of specific RNA-binding proteins comprise three neural specific members called HuD, HuC, and Hel-N1. We have shown that purified recombinant HuD can bind with high affinity to GAP-43 mRNA. In addition, we have mapped the binding site to a highly conserved 26-nucleotide sequence within the regulatory element. The binding of HuD to this site is readily displaced by RNA oligonucleotides encoding other HuD binding sites. We also show that only the first and second RNA binding domains of HuD are required for selective binding to GAP-43 mRNA.

Mice with a deficiency in GH function due to disruption of the GH receptor/binding protein gene (GHR(-/-)) are long lived, insulin sensitive, and obese, whereas mice with excess GH function due to expression of a bovine GH transgene (bGH) are short lived, glucose intolerant, and lean. When challenged with a high-fat (HF) diet, we hypothesized that these mice would be differentially susceptible to diet-induced obesity. To test this hypothesis, GHR(-/-), bGH, and littermate control (WT) mice were fed a HF diet (40% kcal) or a nutrient-matched low-fat diet (9% kcal) for 12 wk. On the HF diet, all mice, regardless of genotype, showed a similar percent weight gain and exhibited a significant increase in percent body fat and the mass of epididymal, retroperitoneal, and sc fat pads. For bGH mice, the increase in adipose tissue was relatively small, compared with the WT or GHR(-/-) mice, suggesting some resiliency, although not immunity, to diet-induced obesity. GHR(-/-) mice, which are relatively obese on a low-fat diet, responded to the dietary challenge in a manner similar to WT controls. With HF feeding, all genotypes experienced an increase in insulin levels and depot-dependent effect of adipose tissue. Together, these results further support a role for GH in energy balance regulation and nutrient partitioning. More importantly, because there were genotype-specific effects of diet, these data stress the importance of diet selection and sampling multiple adipose depots in studies with these mouse models.

Abstract.We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPAtreated ceils were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation-independent mRNA stabilization mechanism.

We have shown previously that GAP-43 gene expression during neuronal differentiation is controlled by selective changes in mRNA stability. This process was found to depend on highly conserved sequences in the 3Ј untranslated region (3Ј UTR) of the mRNA. To map the sequences in the GAP-43 3Ј UTR that mediate this post-transcriptional event, we generated specific 3Ј UTR deletion mutants and chimeras with the ␤-globin gene and measured their half-lives in transfected PC12 cells. Our results indicate that there are two distinct instability-conferring elements localized at the 5Ј and 3Ј ends of the GAP-43 3Ј UTR. Of these destabilizing elements, only the one at the 3Ј end is required for the stabilization of the mRNA in response to treatment with the phorbol ester TPA. This 3Ј UTR element consists of highly conserved uridine-rich sequences and contains specific recognition sites for two neural-specific GAP-43 mRNAbinding proteins. Analysis of the levels of mRNA and protein derived from various 3Ј UTR deletion mutants indicated that all mutants were translated effectively and that differences in gene expression in response to TPA were attributable to changes in GAP-43 mRNA stability. In addition, the phorbol ester was found to affect the binding of specific RNA-binding proteins to the 3Ј UTR of the GAP-43 mRNA. Given that, like the GAP-43 mRNA, its degradation machinery and the GAP-43 mRNAbinding proteins are expressed primarily in neural cells, we propose that these factors may be involved in the posttranscriptional regulation of GAP-43 gene expression during neuronal differentiation.

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