Anderson K D, Morin M A, Beckel-Mitchener A, Mobarak C D, Neve R L, Furneaux H M, Burry R, Perrone-Bizzozero N I
Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque 87131, USA.
J Neurochem. 2000 Sep;75(3):1103-14. doi: 10.1046/j.1471-4159.2000.0751103.x.
We have previously shown that the RNA-binding protein HuD binds to a regulatory element in the growth-associated protein (GAP)-43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP-43-positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F-actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP-43 without changes in levels of other NGF-induced proteins, such as SNAP-25 and tau. In mRNA decay studies, HuD stabilized the GAP-43 mRNA, whereas HuD I+II did not have any effect either on GAP-43 mRNA stability or on the levels of GAP-43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP-43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function.
我们之前已经表明，RNA结合蛋白HuD与生长相关蛋白（GAP）-43 mRNA中的一个调控元件结合，并且这种相互作用涉及其前两个RNA识别基序（RRMs）。在本研究中，我们通过在PC12细胞中过表达人HuD蛋白（pcHuD）或其缺少第三个RRM的截短形式（pcHuD I+II），研究了这种相互作用的功能意义。形态学分析显示，在没有神经生长因子（NGF）的情况下，pcHuD细胞伸出含有GAP-43阳性生长锥的短神经突。这些突起还含有微管蛋白和F-肌动蛋白丝，但未被抗神经丝M蛋白的抗体染色。与这种表型相关的是，pcHuD细胞中GAP-43的水平较高，而其他NGF诱导的蛋白，如SNAP-25和tau的水平没有变化。在mRNA降解研究中，HuD使GAP-43 mRNA稳定，而HuD I+II对GAP-43 mRNA稳定性或GAP-43蛋白水平均无任何影响。同样，pcHuD I+II细胞在没有NGF的情况下没有显示出自发性神经突生长，并且对NGF的反应性生长不足。我们的结果表明，在没有NGF的情况下，HuD足以增加GAP-43基因表达和神经突生长，并且该蛋白中的第三个RRM对该功能至关重要。
