Alternative Splicing of Nonmuscle Myosin Heavy Chains

在脊椎动物中，有超过15种不同的肌球蛋白II亚型，每种亚型都含有不同的肌球蛋白II重链(MHC II)。MHC II亚型多样性是由多个基因以及Pre-mRNA的选择性剪接产生的。以前的研究已经证明了MHC II亚型的细胞类型特异性表达以及MHC II亚型在肌肉和神经组织发育过程中的变化。本研究对NMHC II-A、NMHC II-B和NMHC II-C三个非肌肉型MHC II基因的表达调控机制进行了研究。我们一直在研究NMHC II-A和II-C基因的转录调控，以及NMHC II-B和C基因选择性剪接的组织依赖性调控。在研究NMHC II-B选择性剪接调控机制的过程中，我们发现一个新的RNA结合蛋白家族--RBFox家族在神经元特异性选择性剪接NMHC II-B Pre-mRNA中起着关键作用。在这份报告中，我们重点介绍了剪接调控因子RBFox蛋白。 RBFox蛋白在分子的中心区域含有一个保守的RNA识别基序(RRM)，并与RNA五(六)核苷酸(U)GCAUG特异结合。哺乳动物中的RBFox家族蛋白有三个基因，即RBFOX1、RBFox2和Rbfox3。RBFOX1在脑和横纹肌中表达，而RBFox2在脑和肌肉等多种组织中表达。值得注意的是，Rbfox3的表达仅限于神经组织。经RBFox抗体染色分离的脑细胞生化分析和组织学分析表明，神经元特异性剪接变异体NMHC II-B mRNA的表达水平与RBFox-3的表达水平相关较好，而与RBFox-1或RBFox2的表达水平相关性较差。这些观察表明，Rbfox3有助于神经元特异性剪接NMHC II-B mRNA，尽管大脑表达所有三种RBFox蛋白。接下来，我们将研究扩展到利用两个模型系统研究Rbfox3的生物学功能。首先，我们利用了小鼠胚胎癌细胞P19，这些细胞在维甲酸处理后能够分化为神经细胞。P19细胞的神经元分化可以通过长出的轴突样延伸来监测，其中包含轴突标记--磷酸化的神经丝。在神经元分化过程中，Rbfox3被诱导表达，而未分化的P19细胞不表达Rbfox3。在未分化和分化条件下，RBFOX1几乎检测不到，分化前后RBFox2表达水平无明显变化。ShRNA介导的Rbfox3基因敲除导致轴突样伸展减少，几乎完全消除了磷酸化的神经细丝。这些结果表明，Rbfox3是P19细胞向神经元分化所必需的。其次，我们利用鸡胚胎脊髓来研究Rbfox3在体内神经元发育中的作用。SiRNA介导的功能丧失研究表明，Rbfox3需要促进有丝分裂后运动神经元和中间神经元的神经元分化。编码信号转导蛋白的Numb-Pre-mRNA被发现是Rbfox3作用的靶标，Rbfox3通过与上游内含子中保守的UGCAUG元件结合来抑制另一个外显子的包含。通过siRNA耗尽特定数量剪接异构体会复制类似的神经元分化缺陷。相关数字剪接异构体的强制表达足以以同种形式特异性的方式将有丝分裂后神经元从Rbfox3缺失导致的分化缺陷中拯救出来。因此，Rbfox3依赖的Numb选择性剪接在脊椎动物发育过程中的神经元分化过程中起着重要作用。

In vertebrates, there are over 15 different myosin II isoforms, each of which contains different myosin II heavy chains (MHC IIs). MHC II isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC II isoforms as well as changes in MHC II isoforms during muscle and neural tissue development. This research program has investigated the regulatory mechanisms responsible for the expression of three nonmuscle MHC II (NMHC II) genes, NMHC II-A, NMHC II-B, and NMHC II-C. We have been studying the transcriptional regulation of NMHC II-A and II-C genes as well as tissue-dependent regulation of alternative splicing of NMHC II-B and C genes. During the course of studying the regulatory mechanism for alternative splicing of NMHC II-B, we found that a new RNA binding protein family, the Rbfox family, plays a critical role for neuron-specific alternative splicing of NMHC II-B pre-mRNA. In this report, we focus on the splicing regulators, Rbfox proteins. Rbfox proteins contain a single conserved RNA recognition motif (RRM) in the central region of the molecule and bind specifically to an RNA penta(hexa)nucleotide (U)GCAUG. There are three genes for Rbfox family proteins in mammals, Rbfox1, Rbfox2 and Rbfox3. Rbfox1 is expressed in brain and striated muscles whereas Rbfox2 is expressed in various tissues including brain and muscles. Notably, Rbfox3 expression is restricted to neural tissues. Biochemical analyses of brain cells sorted by Rbfox antibody staining and histological analyses demonstrated that the expression level of the neuron-specific splice variant of NMHC II-B mRNA correlated better with the level of Rbfox-3 expression rather than with that of Rbfox-1 or Rbfox2 expression. These observations suggest that Rbfox3 contributes neuron-specific splicing of NMHC II-B mRNA, although brain expresses all three Rbfox proteins. Next we extended our research to study the biological function of Rbfox3 using two model systems. First we made use of mouse embryonic carcinoma P19 cells which are capable of differentiating into neuronal cells following retinoic acid treatment. Neuronal differentiation of P19 cells can be monitored by outgrowth of a long axon-like extension which contains an axonal marker, phosphorylated neurofilaments. During neuronal differentiation, expression of Rbfox3 is induced whereas undifferentiated P19 cells do not express Rbfox3. Rbfox1 is barely detected under both undifferentiated and differentiated conditions and the Rbfox2 expression level is unchanged before and after differentiation. The shRNA-mediated knock-down of Rbfox3 results in a decrease in axon-like extensions and an almost complete elimination of phosphorylated neurofilaments. These results indicate that Rbfox3 is required for neuronal differentiation of P19 cells. Second we used the chicken embryonic spinal cord to study a role for Rbfox3 in neuronal development in vivo. SiRNA-mediated loss-of-function studies show that Rbfox3 is required to promote neuronal differentiation of postmitotic motor neurons as well as interneurons. Numb pre-mRNA encoding a signaling adaptor protein is found to be a target of Rbfox3 action, and Rbfox3 represses the inclusion of an alternative exon via binding to the conserved UGCAUG element in the upstream intron. Depleting a specific Numb splice isoform by siRNA reproduces similar neuronal differentiation defects. Forced expression of the relevant Numb splice isoform is sufficient to rescue, in an isoform-specific manner, postmitotic neurons from defects in differentiation caused by Rbfox3 depletion. Thus, Rbfox3-dependent Numb alternative splicing plays an important role in the progression of neuronal differentiation during vertebrate development.