DLP，一个新的DIM1蛋白家族成员—参与Pre-mRNA剪接与细胞周期调控
摘要
发现新的功能基因，确定每个基因的独特功能和生物学意义，并阐明其调节机制，是功能基因组时代研究的一个重要领域。阐明基因选择性表达所依赖的调控信息及其相互作用的分子机制，是揭示生命现象本质的核心问题，是结构基因组之后功能基因组研究的重要内容。随着基因组学的广泛开展，基因的表达调控研究已经逐渐从单个基因点、线式的调控拓展到立体层面上多基因、基因簇以至整个基因组的调控网络。如何有效利用已有的基因组学数据，充分整合多学科的新思路，并建立新的实验系统和技术体系，阐明真核基因组表达的调控网络，已经成为功能基因组学领域内国际竞争的焦点。因此，识别出人类基因组中所有的基因并对其功能进行分析成为生物医学研究的重点之一。尤其是对细胞因子、原癌基因、转录因子、剪接因子和信号传导因子等的克隆和鉴定。同时生物信息学技术的发展也为发现新的，低丰度基因提供了可行性。
我们在对一个细胞因子类似因子-CKLF的研究过程中，发现在其染色体区域附近约10Mb处存在一个新的蛋白质编码基因。对这个基因进行生物信息学分析，没有得到有关趋化因子的提示，这个基因的结构分析表明从4-135氨基酸为一个DIMl结构域，因此我们将这个基因编码的蛋白称为DIM1-like Protein(DLP)。氨基酸序列进行同源性分析发现DLP与酵母的一个15KD的剪接体蛋白U5有弱的同源性，和一种有丝分裂蛋白DIMl及人硫氧还蛋白分别有39％、24％的同源性，同时发现它在人、鼠、Arabidopsis thaliana、Oryza、melanogaster、果蝇、酵母菌等各种属之间有相当的保守性。DLP的序列与Golemis命名的DIM2相同(Zhang et al．1999)。因此，我们推测DLP也许是DIM1家族的成员之一。向GenBank提交登记获得接受，GenBank的登记号为AY566808。DIM1属近几年发现的一个硫氧还原蛋白超家族的第六分支。在人和酵母中，DIM1蛋白是U4／U6和U5tri-snRNP的一个亚基，其突变能使细胞停滞在G2／M期并且影响Pre-mRNA剪接过程。考虑到DIM1蛋白可能是通过影响Pre-mRNA的剪接来间接影响细胞周期，所以我们设想DLP也可能参与Pre-mRNA剪接的过程。
我们首先对DLP进行表达谱分析，Nothem Blot显示DLP在骨骼肌、肝脏、心脏和胰腺中高表达，在肾脏，脑和胎盘中中等程度的表达，在肺中表达较低。RT-PCR证实DLP在人的乳腺癌细胞MCF-7，肝癌细胞HepG2和子宫内膜癌细胞Ishikawa中也有高表达。说明DLP在体内多个组织均有表达但又有特异性。用绿色荧光蛋白做定位表明DLP
Metazoans display an extraordinarily broad spectrum of functional and behavioural complexity. The size of the proteome of an organism can be expanded during evolution by increasing the numbers of genes, by elaborating pre-existing mechanisms that generate protein diversity and/or by elaborating the relationships between components (for example, regulatory networks), and by inventing new mechanisms. Mechanisms that increase protein diversity in all metazoans include the use of multiple transcription start sites, alternative pre-mRNA splicing, polyadenylation, pre-mRNA editing, and post-transcriptional protein modification. Among these mechanisms, alternative Pre-mRNA splicing is considered to be the most important source of protein diversity in vertebrates. The recent completion of a draft of human genome indicated that alternative splicing of Pre-mRNAs plays a crucial role in obtaining biological complexity, because more than 59% of the human genes seem to be alternatively spliced (Hastings and Krainer 2001).In addition, it was estimated that about 15% of all mutations that cause a genetic disease in humans lead to a failure in the correct splicing of the corresponding pre-mRNA, which disturbs the expression of the gene (Philips and Cooper 2000). Thus, the removal of pre-mRNA introns plays a major role in gene regulation. As of 1999, around 100 splicing factors were identified; however (Burge et al., 1999), that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures.
Both genetic and biochemical studies have revealed that essential components of the spliceosome include five small RNAs-U1, U2, U4, U5 and U6, and as many as 300 distinct proteins. Here we reported the molecular cloning and functional analysis of a novel cDNA encoding for a protein of 149 amino acids. This protein has 38% amino acid sequence identity
我们在对一个细胞因子类似因子-CKLF的研究过程中，发现在其染色体区域附近约10Mb处存在一个新的蛋白质编码基因。对这个基因进行生物信息学分析，没有得到有关趋化因子的提示，这个基因的结构分析表明从4-135氨基酸为一个DIMl结构域，因此我们将这个基因编码的蛋白称为DIM1-like Protein(DLP)。氨基酸序列进行同源性分析发现DLP与酵母的一个15KD的剪接体蛋白U5有弱的同源性，和一种有丝分裂蛋白DIMl及人硫氧还蛋白分别有39％、24％的同源性，同时发现它在人、鼠、Arabidopsis thaliana、Oryza、melanogaster、果蝇、酵母菌等各种属之间有相当的保守性。DLP的序列与Golemis命名的DIM2相同(Zhang et al．1999)。因此，我们推测DLP也许是DIM1家族的成员之一。向GenBank提交登记获得接受，GenBank的登记号为AY566808。DIM1属近几年发现的一个硫氧还原蛋白超家族的第六分支。在人和酵母中，DIM1蛋白是U4／U6和U5tri-snRNP的一个亚基，其突变能使细胞停滞在G2／M期并且影响Pre-mRNA剪接过程。考虑到DIM1蛋白可能是通过影响Pre-mRNA的剪接来间接影响细胞周期，所以我们设想DLP也可能参与Pre-mRNA剪接的过程。
我们首先对DLP进行表达谱分析，Nothem Blot显示DLP在骨骼肌、肝脏、心脏和胰腺中高表达，在肾脏，脑和胎盘中中等程度的表达，在肺中表达较低。RT-PCR证实DLP在人的乳腺癌细胞MCF-7，肝癌细胞HepG2和子宫内膜癌细胞Ishikawa中也有高表达。说明DLP在体内多个组织均有表达但又有特异性。用绿色荧光蛋白做定位表明DLP
Metazoans display an extraordinarily broad spectrum of functional and behavioural complexity. The size of the proteome of an organism can be expanded during evolution by increasing the numbers of genes, by elaborating pre-existing mechanisms that generate protein diversity and/or by elaborating the relationships between components (for example, regulatory networks), and by inventing new mechanisms. Mechanisms that increase protein diversity in all metazoans include the use of multiple transcription start sites, alternative pre-mRNA splicing, polyadenylation, pre-mRNA editing, and post-transcriptional protein modification. Among these mechanisms, alternative Pre-mRNA splicing is considered to be the most important source of protein diversity in vertebrates. The recent completion of a draft of human genome indicated that alternative splicing of Pre-mRNAs plays a crucial role in obtaining biological complexity, because more than 59% of the human genes seem to be alternatively spliced (Hastings and Krainer 2001).In addition, it was estimated that about 15% of all mutations that cause a genetic disease in humans lead to a failure in the correct splicing of the corresponding pre-mRNA, which disturbs the expression of the gene (Philips and Cooper 2000). Thus, the removal of pre-mRNA introns plays a major role in gene regulation. As of 1999, around 100 splicing factors were identified; however (Burge et al., 1999), that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures.
Both genetic and biochemical studies have revealed that essential components of the spliceosome include five small RNAs-U1, U2, U4, U5 and U6, and as many as 300 distinct proteins. Here we reported the molecular cloning and functional analysis of a novel cDNA encoding for a protein of 149 amino acids. This protein has 38% amino acid sequence identity
引文
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