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。本研究计划研究了两个非肌肉MHC II(NMHC II)基因NMHC II-B和NMHC II-C的组织依赖选择性剪接的调控机制。 我们过去用培养细胞进行的研究表明，RNA结合蛋白家族RBFox家族在NMHC II-B前-mRNA的神经元特异性选择性剪接中发挥作用。RBFox蛋白在分子的中心区域含有一个保守的RNA识别基序，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。通过RBFox抗体染色和组织学分析分选的小鼠脑细胞的生化分析表明，神经元特异性剪接变异体NMHC II-B mRNA的表达水平与Rbfox3的表达水平相关联，而不是与RBFox-1或RBFox2的表达水平相关，尽管Rbfox3及其部分RBFOx1和2的亚型在培养细胞中过表达时类似地能够促进神经元特异性外显子B1的剪接。这些观察表明，Rbfox3在体内NMHC II-B mRNA的神经元特异性剪接中起着更具生理学意义的作用。为了了解Rbfox3介导的选择性剪接调控机制，我们搜索了与Rbfox3相互作用的核因子(S)。我们通过抗体亲和层析从小鼠脑提取物中提取含Rbfox3的复合体，确定了PTB相关剪接因子(PSF，也称为SFPQ)是一种与Rbfox3相互作用的蛋白。Rbfox3的C-末端区域直接与PSF的N-末端区域结合。在培养细胞中，Rbfox3对B1包涵体的增强依赖于PSF的存在。Rbfox3以PSF依赖的方式被招募到内源NMHC II-B转录本B1下游的UGCAUG元件。PSF以UGCAUG依赖的方式增强B1的包含性，尽管它不直接与该元件结合。因此，PSF作为Rbfox3的重要共激活剂发挥作用。Rbfox3和PSF的相互作用是RBFox蛋白通过下游内含子增强子调节替代外显子激活的机制的组成部分。我们现在正在扩展我们的研究，以研究NMHC II-C mRNAs中替代外显子C1的肌肉特异性跳过。小鼠成肌细胞系C2C12和G8提供了有用的模型，在该模型中，在从成肌细胞向多核肌管分化过程中，排除C1的NMHC II-C mRNA剪接变体的表达水平高于包含C1的变体的表达水平。使用包含C1外显子和侧翼内含子和外显子的微型基因构建物，尽管在C1的上游和下游内含子中都有多个UGCAUG元件，但发现C1上游的近端UGCAUG元件对于mRNAs中的C1排除是重要的。在成肌细胞中过表达RBFOX1和2可通过上游UGCAUG增强C1排斥。这与RBFox激活的B1的包含相反，后者需要下游的UGCAUG元件。这些结果表明，RBFox蛋白具有选择性剪接的双重功能：激活或抑制替代外显子，可能是通过不同的机制。

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 tissue-dependent alternative splicing of two nonmuscle MHC II (NMHC II) genes, NMHC II-B and NMHC II-C. Our past study using cultured cells revealed that an RNA binding protein family, the Rbfox family, plays a role for neuron-specific alternative splicing of NMHC II-B pre-mRNA. Rbfox proteins contain a single conserved RNA recognition motif 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 mouse 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 Rbfox3 expression rather than with that of Rbfox-1 or Rbfox2 expression, although Rbfox3 and some of the isoforms of Rbfox1 and 2 are similarly capable of enhancing the neuron-specific exon B1 splicing when they are over-expressed in cultured cells. These observations suggest that Rbfox3 plays a more physiologically relevant role in neuron-specific splicing of NMHC II-B mRNA in vivo. To understand a mechanism for Rbfox3-mediated regulation of alternative splicing, we searched for nuclear factor(s) which interact with Rbfox3. We identified PTB-associated splicing factor (PSF, also called SFPQ) as an interacting protein with Rbfox3 by antibody affinity-chromatography of Rbfox3-containing complexes from mouse brain extracts. The C-terminal region of Rbfox3 directly binds to the N-terminal region of PSF. In cultured cells, enhancement of B1 inclusion by Rbfox3 depends on the presence of PSF. Rbfox3 is recruited to the UGCAUG element downstream of B1 in the endogenous NMHC II-B transcript in a PSF-dependent manner. PSF enhances B1 inclusion in a UGCAUG-dependent manner, although it does not bind directly to this element. Therefore, PSF functions as an essential co-activator of Rbfox3. Rbfox3 and PSF interaction is an integral part of the mechanism by which Rbfox proteins regulate activation of alternative exons via a downstream intronic enhancer. We are now extending our research to study muscle-specific skipping of the alternative exon C1 in NMHC II-C mRNAs. Mouse myogenic cell lines C2C12 and G8 provide useful models in which the expression level of the C1-excluded NMHC II-C mRNA splice variant over the C1-included variant increases during differentiation from myoblasts to multinucleated myotubes. Using minigene constructs containing the C1 exon and flanking introns and exons, a proximal UGCAUG element upstream of C1 was found to be important for C1 exclusion in mRNAs, although there are multiple UGCAUG elements in both upstream and downstream introns of C1. Overexpression of either Rbfox1 and 2 in myoblasts enhances C1 exclusion via the upstream UGCAUG. This is in contrast with Rbfox-activated inclusion of B1 which requires the downstream UGCAUG element. These results suggest that Rbfox proteins have dual functions for alternative splicing: to either activate or to repress an alternative exon, presumably by different mechanisms.