Alternative Splicing of Nonmuscle Myosin Heavy Chains

非肌肉肌球蛋白重链的选择性剪接

• 批准号: 9557299
• 负责人: Robert Adelstein
• 金额: $ 51.71万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9557299
• 关键词: Affinity Chromatography; Alternative Splicing; Amino Acids; Antibodies; Antibody Affinity; Binding; Biochemical; Biological Models; Brain; C-terminal; Cell Line; Cell physiology; Complex; Cultured Cells; Development; Elements; Enhancers; Exclusion; Exons; Family; Genes; Histologic; Introns; Mammals; Mediating; Messenger RNA; Modeling; Mus; Muscle; Muscle Cells; Muscle Fibers; Myoblasts; Myosin Heavy Chains; Myosin Type II; N-terminal; Neurons; Nonmuscle Myosin Type IIB; Nuclear; Nucleotides; Pentas; Physiological; Play; Protein Family; Protein Isoforms; Proteins; RNA; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; Recruitment Activity; Regulation; Research; Role; Staining method; Stains; Striated Muscles; Tissues; Transcript; Variant; Vertebrates; brain cell; cell type; in vivo; mRNA Precursor; non-muscle myosin; overexpression; paralogous gene; programs; relating to nervous system

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.

在脊椎动物中，有超过 15 种不同的肌球蛋白 II 亚型，每种亚型都包含不同的肌球蛋白 II 重链 (MHC II)。 MHC II 同工型多样性是由多个基因以及前 mRNA 的选择性剪接产生的。先前的研究已经证明了 MHC II 同工型的细胞类型特异性表达以及 MHC II 同工型在肌肉和神经组织发育过程中的变化。该研究项目研究了两个非肌肉 MHC II (NMHC II) 基因 NMHC II-B 和 NMHC II-C 的组织依赖性选择性剪接的调节机制。 我们过去使用培养细胞的研究表明，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 蛋白在分子中心区域包含一个保守的 RNA 识别基序，并与 RNA 五（六）核苷酸 (U)GCAUG 特异性结合。哺乳动物中的 Rbfox 家族蛋白有 3 个基因：Rbfox1、Rbfox2 和 Rbfox3。 Rbfox1 在大脑和横纹肌中表达，而 Rbfox2 在包括大脑和肌肉在内的各种组织中表达。值得注意的是，Rbfox3 表达仅限于神经组织。通过 Rbfox 抗体染色分选的小鼠脑细胞的生化分析和组织学分析表明，NMHC II-B mRNA 神经元特异性剪接变体的表达水平与 Rbfox3 表达水平相关性更好，而不是与 Rbfox-1 或 Rbfox2 表达水平相关性更好尽管 Rbfox3 以及 Rbfox1 和 2 的一些亚型同样能够增强神经元特异性外显子 B1 剪接当它们在培养细胞中过度表达时。这些观察结果表明，Rbfox3 在体内 NMHC II-B mRNA 的神经元特异性剪接中发挥着更生理学相关的作用。为了了解 Rbfox3 介导的选择性剪接调节机制，我们寻找与 Rbfox3 相互作用的核因子。我们通过对小鼠脑提取物中含有 Rbfox3 的复合物进行抗体亲和层析，将 PTB 相关剪接因子（PSF，也称为 SFPQ）鉴定为与 Rbfox3 相互作用的蛋白。 Rbfox3 的 C 端区域直接与 PSF 的 N 端区域结合。在培养细胞中，Rbfox3 对 B1 包涵体的增强取决于 PSF 的存在。 Rbfox3 以 PSF 依赖性方式被招募至内源性 NMHC II-B 转录物中 B1 下游的 UGCAUG 元件。 PSF 以 UGCAUG 依赖性方式增强 B1 包含，尽管它不直接与该元件结合。因此，PSF 是 Rbfox3 的重要共激活剂。 Rbfox3 和 PSF 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一个组成部分。我们现在正在扩展我们的研究，以研究 NMHC II-C mRNA 中替代外显子 C1 的肌肉特异性跳跃。小鼠肌原细胞系 C2C12 和 G8 提供了有用的模型，其中 C1 排除的 NMHC II-C mRNA 剪接变体的表达水平在从成肌细胞分化为多核肌管的过程中相对于包含 C1 的变体有所增加。使用包含 C1 外显子和侧翼内含子和外显子的小基因构建体，发现 C1 上游的近端 UGCAUG 元件对于 mRNA 中的 C1 排除很重要，尽管 C1 的上游和下游内含子中都有多个 UGCAUG 元件。成肌细胞中 Rbfox1 和 2 的过度表达可通过上游 UGCAUG 增强 C1 排斥。这与 Rbfox 激活的 B1 内含物形成对比，后者需要下游 UGCAUG 元件。这些结果表明，Rbfox 蛋白具有选择性剪接的双重功能：可能通过不同的机制激活或抑制替代外显子。