Multiple novel nesprin-1 and nesprin-2 variants act as versatile tissue-specific intracellular scaffolds.
Multiple novel nesprin-1 and nesprin-2 variants act as versatile tissue-specific intracellular scaffolds.
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DOI：
10.1371/journal.pone.0040098
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Shanahan CM
中科院分区：
文献类型：
--
作者：
Rajgor D;Mellad JA;Autore F;Zhang Q;Shanahan CM
核包膜光影蛋白重复蛋白(Nesprs)是一个新的含有光影蛋白重复序列的蛋白家族。Nesprin-1和Nesprin-2基因由146个外显子和116个外显子组成，分别编码∼1 mDa和∼800 kDa的蛋白。然而，新出现的数据表明，尼斯普林斯在其基因中有多个可供选择的起始和终止位点，从而允许产生更小的异构体。在这项研究中，我们开始通过筛选EST数据库和使用RACE分析来识别cDNA端来鉴定新的可选择转录的Nesprin变异体。这两种方法为可供选择的起始和终止位点提供了潜在的命中，并通过聚合酶链式反应和DNA测序进行了验证。我们发现，这些替代位点不仅以组织特异性的方式表达，而且通过将不同的位点结合在一起，有可能创造出广泛的Nesprin变体。通过克隆和在人成纤维细胞和U2OS细胞中表达新的Nesprin变异体，我们显示了肌动蛋白应力纤维、局灶性粘连、微管、核仁、核基质和核膜(NE)的定位。此外，我们还表明，不同的细胞类型不同，单个Nesprin变异体的亚细胞定位也不同，这表明任何一个Nesprin变异体在不同的细胞类型中可能具有不同的功能。这些研究表明，尼斯普林斯是高度通用的组织特异性细胞内蛋白支架，并确定了尼斯普林斯超越细胞质-核耦合的潜在新功能。这些交替的功能也可能解释了当这些基因突变时观察到的不同疾病表型范围。
Nesprins (Nuclear envelope spectrin-repeat proteins) are a novel family of giant spectrin-repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of ∼1mDa and ∼800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms. In this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types. These studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account for the diverse range of disease phenotypes observed when these genes are mutated.
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