Abstract
The neuronal ELAV-like RNA-binding protein HuD binds to a regulatory element in the 3'-untranslated region of the growth-associated protein-43 (GAP-43) mRNA. Here we report that overexpression of HuD protein in PC12 cells stabilizes the GAP-43 mRNA by delaying the onset of mRNA degradation and that this process depends on the size of the poly(A) tail. Using a polysome-based in vitro mRNA decay assay, we found that addition of recombinant HuD protein to the system increased the half-life of full-length, capped, and polyadenylated GAP-43 mRNA and that this effect was caused in part by a decrease in the rate of deadenylation of the mRNA. This stabilization was specific for GAP-43 mRNA containing the HuD binding element in the 3'-untranslated region and a poly(A) tail of at least 150 A nucleotides. In correlation with the effect of HuD on GAP-43 mRNA stability, we found that HuD binds GAP-43 mRNAs with long tails (A150) with 10-fold higher affinity than to those with short tails (A30). We conclude that HuD stabilizes the GAP-43 mRNA through a mechanism that is dependent on the length of the poly(A) tail and involves changes in its affinity for the mRNA.
Highlights
The growth-associated protein growth-associated protein-43 (GAP-43) is a neuronal-specific phosphoprotein that is expressed primarily in neurons during both the initial axonal outgrowth and remodeling of synaptic connections for reviews [1,2,3]
We have previously shown that HuD stabilizes the GAP-43 mRNA in transfected PC12 cells and neurons, leading to increased protein expression and neuronal differentiation [18, 19, 30]
The pMEP4GAP vector allows the transient expression of the GAP-43 mRNA by cadmium induction; its subsequent decay can be measured by Northern blot analysis in the absence of any transcriptional inhibitors [10, 16]
Summary
The growth-associated protein GAP-43 is a neuronal-specific phosphoprotein that is expressed primarily in neurons during both the initial axonal outgrowth and remodeling of synaptic connections for reviews [1,2,3]. We report that overexpression of HuD protein in PC12 cells stabilizes the GAP-43 mRNA by delaying the onset of mRNA degradation and that this process depends on the size of the poly(A) tail.
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