CHARACTERIZATION OF NEURONAL CELL-SPECIFIC RNA-BINDING PROTEINS IN THE DEVELOPMENT OF RETINA

神经元RNA结合蛋白在选择性剪接、RNA转运和局部翻译等基因表达的转录后调控中发挥重要作用。到目前为止，已经描述了几个神经元rna结合蛋白家族。例如，Hu蛋白已被确定为与肺小细胞癌相关的人副肿瘤脑神经病变的自身抗原。Hu蛋白的主要功能之一是通过结合RNA稳定元件调节mRNA代谢。Nova-1，被称为副肿瘤性opsoclonus-myoclonus共济失调抗原，也是一种调节选择性剪接的神经元特异性RNA结合蛋白。最近，我们从人类和大鼠cDNA文库中克隆了一个PTB同源物，作为癌症相关视网膜病变的可能自身抗原。这种新蛋白被命名为ptb样蛋白（PTBLP）。在本研究中，我们用PC12细胞和PTBLP缺失的小鼠研究了PTBLP在神经元分化中的可能作用。在ngf诱导的PC12细胞神经元分化过程中，PTBLP-L（长链）表达下调。PTBLP-L转染PC12细胞后，神经元分化受到抑制。然而，在PTBLP-S（简称，缺乏RNA结合能力）转染的细胞中，这种抑制并不明显。当PTBLP-L和PTBLP-S同时转染时，PTBLP-L的抑制作用减弱。在分化的细胞中，PTBLP-S定位于细胞核，PTBLP-L分散在细胞质和神经元生长锥中。这些发现表明PTBLP-L是神经元分化的负调节因子，而PTBLP-S是PTBLP-L的竞争对手。为了探索PTBLP在完整动物体内的功能，我们采用同源重组的方法敲除小鼠PTBLP基因。PTBLP缺失小鼠在胚胎后期死亡。PTBLP杂合子未见表型异常。这些结果提示PTBLP可能在神经元发育中起重要作用。少

Neuronal RNA-binding proteins play an important role in post-transcriptional regulation of gene expression such as alternative splicing, RNA transport and local translation. Several families of neuronal RNA-binding proteins have been so far described. For example, Hu proteins have been identified as an autoantigen of human paraneoplastic encephaloneuropathies associated with lung small cell carcinomas. One of the major functions of Hu proteins is regulation of mRNA metabolism through binding to RNA stability elements. Nova-1, known as the paraneoplastic opsoclonus-myoclonus ataxia antigen, is also a neuron-specific RNA binding protein that regulates alternative splicing. Recently, we have cloned a homologue of PTB as a possible autoantigen of cancer-associated retinopathy from human and rat cDNA libraries. This new protein was named as PTB-like protein (PTBLP).In this study, the possible roles of PTBLP on neuronal differentiation were examined using PC12 cells and PTBLP null mice. Duri … More ng NGF-induced neuronal differentiation in PC12 cells, PTBLP-L (long form) was down-regulated. By transfection of PTBLP-L into PC12 cells, the neuronal differentiation was suppressed. In PTBLP-S (short form ; lack the RNA binding ability) transfected cells, however, this suppression was not evident. When both PTBLP-L and PTBLP-S were co-transfected, the suppressive effect of PTBLP-L decreased. In differentiated cells, PTBLP-S localized in the nucleus and PTBLP-L was found dispersed throughout the cytoplasm and neuronal growth cone. These findings suggest that PTBLP-L acts as a negative regulator of neuronal differentiation and PTBLP-S acts as a competitor of PTBLP-L. To explore the functions of PTBLP in the intact animal, we undertook to knock out the PTBLP gene in mice by homologous recombination. PTBLP null mice were died in embryonic late stage. There was no abnormal phenotype in PTBLP heterozygotes. Those results are suggested that PTBLP may play an important role in neuronal development. Less