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。在这份报告中，我们重点关注NMHC II-B的选择性剪接的调控。 编码NMHC II-B的基因产生交替剪接的异构体，包括或排除ATP结合域附近的一盒氨基酸(AA)。在NMHC II-B mRNAs中，编码10个氨基酸的选择性外显子B1(也称为外显子N30)仅限于某些类型的神经细胞。我们以前已经报道过B1下游的内含子区域，称为内含子远端下游增强子(IDDE)，是激活B1剪接所必需的。IDDE中的RNA元件UGCAUG的两个副本对于神经细胞中的B1包涵体是必不可少的。最近，有报道称线虫Fox-1的一个脊椎动物同源物以高度序列特异性的方式与UGCAUG结合。数据库搜索显示，哺乳动物中存在Fox-1同源基因(Fox家族)，Fox-1(又称A2BP1)，Fox-2(又称Fxh和Rbm9)和Fox-3。Fox-1在脑和横纹肌中表达，而Fox-2在包括脑和肌肉在内的各种组织中表达。值得注意的是，Fox-3的表达仅限于神经组织。我们提出了针对每种Fox蛋白的抗体，并对Fox蛋白在小鼠脑和脊髓中的分布进行了组织学分析。Fox-1、Fox-2和Fox-3在多种神经细胞中表达重叠，但我们也观察到它们在某些类型神经细胞中的表达差异。将这些Fox表达模式与原位杂交检测到的包含B1的NMHC II-B mRNA的表达进行比较，表明Fox-3表达的细胞倾向于包含B1插入片段。对大脑和脊髓细胞B1剪接模式的RT-PCR分析进一步支持了这一观点，这些剪接模式是根据Fox-3的表达分离和分类的。来自小脑、脑干和脊髓的Fox-3阳性细胞以浓度依赖的方式包括B1插入片段。尽管Fox-3阴性细胞表达Fox-1和Fox-2，但几乎完全排除了B1插入。因此，尽管Fox-3和Fox-1和Fox-2的一些亚型在培养细胞中过表达时，B1的包含程度与Fox-3的表达水平相比与Fox-1或Fox-2的表达水平有更好的相关性，但它们类似地能够促进B1的剪接。 为了了解FOX介导的选择性剪接调控机制，我们搜索了与FOX-3相互作用的核因子(S)。我们通过亲和层析鉴定了PTB相关剪接因子(PSF)是一种与Fox-3相互作用的蛋白。Fox-3的C-末端区域与PSF的N-末端区域直接结合。在培养细胞中，Fox-3对B1包涵体的增强依赖于PSF的存在。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。FOX-3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。因此，PSF作为FOX-3的重要共激活因子发挥作用。FOX-3和PSF的相互作用是FOX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。

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 the course of 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. In this report, we focus on regulation of alternative splicing of NMHC II-B. The gene encoding NMHC II-B generates alternatively spliced isoforms, which include or exclude a cassette of amino acids (aa) near the ATP-binding domain. Inclusion of alternative exon B1 (also called exon N30) encoding 10 aa in NMHC II-B mRNAs is restricted to some types of neural cells. We have previously reported that the intronic region downstream of B1, called the intronic distal downstream enhancer (IDDE), is required for activation of B1 splicing. Two copies of an RNA element UGCAUG within the IDDE are essential for B1 inclusion in neural cells. Recently a vertebrate homolog of C. elegans Fox-1 was reported to bind to UGCAUG in a highly sequence-specific manner. Database search revealed that there are three genes for Fox-1 homologs (Fox family) in mammals, Fox-1 (also called A2BP1), Fox-2 (also called Fxh and Rbm9) and Fox-3. Fox-1 is expressed in brain and striated muscles whereas Fox-2 is expressed in various tissues including brain and muscles. Notably, the Fox-3 expression is restricted to neural tissues. We raised antibodies specific to each of the Fox proteins and histologically analyzed distribution of Fox proteins in mouse brain and spinal cord. Expressions of Fox-1, 2 and 3 overlap in many kinds of neural cells, but we also observed differences in their expression in certain types of neural cells. Comparison of these Fox expression patterns with expression of the B1-included NMHC II-B mRNA detected by in situ hybridization suggested that the Fox-3 expressing cells tend to include the B1 insert. This notion was further supported by the RT-PCR analysis of B1 splicing patterns of brain and spinal cord cells which were dissociated and sorted according to Fox-3 expression. Fox-3 positive cells from cerebellum, brainstem and spinal cord include the B1 insert in a Fox-3 concentration-dependent manner. Fox-3 negative cells almost completely exclude the B1 insert, despite expression of Fox-1 and 2. Therefore there is a better correlation of the extent of B1 inclusion with the level of Fox-3 expression rather than with that of Fox-1 or 2 expression in intact tissues, although Fox-3 and some of the isoforms of Fox-1 and 2 are similarly capable of enhancing B1 splicing when they are over-expressed in cultured cells. To understand a mechanism for Fox-mediated regulation of alternative splicing, we searched for nuclear factor(s) which interact with Fox-3. We identified PTB-associated splicing factor (PSF) as an interacting protein with Fox-3 by affinity-chromatography. The C-terminal region of Fox-3 directly binds to the N-terminal region of PSF. In cultured cells, enhancement of B1 inclusion by Fox-3 depends on the presence of PSF. PSF enhances B1 inclusion in a UGCAUG-dependent manner, although it does not bind directly to this element. Fox-3 is recruited to the UGCAUG element downstream of B1 in the endogenous NMHC II-B transcript in a PSF-dependent manner. Therefore PSF functions as an essential co-activator of Fox-3. Fox-3 and PSF interaction is an integral part of the mechanism by which Fox proteins regulate activation of alternative exons via a downstream intronic enhancer.