Tra2β1蛋白在神经特异性的基因剪接中的功能

体外(in vitro)生化研究已证明哺乳动物Tra2蛋白是前体mRNA剪接的重要调控因子,但是,对该蛋白在in vivo条件下的剪接功能,尤其是在神经特异性基因剪接中的功能及其细胞特异性,目前所知甚少。本文采用in vivo分析模型,在COS-1和PFSK两种不同类型的细胞中,研究了两个神经特异性基因(GluR-B,SMN2)剪接的细胞特异性,同时分析了Tra2β1在这两个基因剪接中的功能及其细胞特异性。结果表明,在研究的两种细胞中,GluR-B和SMN2“小基因”的剪接均具有明显的细胞特异性;而Tra2β1蛋白的过量表达在这两种不同的细胞中对“小基因”的剪接有显著的相同倾向的调节作用,提示Tra2β1蛋白对该两个基因剪接的调节作用可能没有细胞特异性。     

前体mRNA剪接及剪接调节因子SR蛋白和Tra2蛋白

在高等真核生物中,前体mRNA的剪接及其调节是一个复杂的、由多因子参与的过程,它对基因的正常功能的发挥起着重要的作用,任何一种剪接调节因子的异常变化均有可能导致疾病的发生。因此,研究参与前体mRNA剪接调控的相关因子的功能及作用机制,对前体mRNA剪接机制的阐明,无疑是相当必要的。本文着重介绍了两类重要的mRNA剪接调节蛋白——SR蛋白和Tra2蛋白的研究近况,以期对前体mRNA剪接机制的研究的重要性和复杂性有更多的了解。     

前体mRNA剪接蛋白Tra2β1的抗体制备及鉴定

Tra2 β1是Tra2 β前体mRNA剪接调节蛋白家族中的一个组织分布最广、表达量最多的成员 ,它在选择性前体mRNA剪接中有调节功能 ,而在基础性剪接中不是必需的 .为便于对该蛋白功能的进一步研究 ,需要制备能特异地检测Tra2 β1蛋白的抗体 .选择包含Tra2 β1的N端 12个氨基酸的特异编码序列的Tra2 β2全长编码序列 (共 117个核苷酸 ) ,将其克隆至pGEX 3X表达载体形成GST融合基因 ,并以诱导表达和纯化的该融合蛋白为抗原免疫新西兰兔 ,获得了相应的抗体 .Western印迹和免疫细胞化学分析结果显示 ,获得的抗体能特异地检测Tra2 β1蛋白     

U2AF65蛋白表达水平影响基因UBQLN1的可变剪接

目的:研究U2AF65蛋白的表达水平对基因UBQLN1可变剪接的影响。方法:应用pSR-GFP/Neo载体构建2个U2AF65-siRNA干扰载体,转染293T细胞,通过Western印迹、QRT-PCR检测干扰效果,RT-PCR验证基因UBQLN1的可变剪接。结果:利用设计的U2AF65-siRNA能够干扰细胞中U2AF65的表达;RT-PCR结果显示U2AF65表达水平的下降促使UBQLN1第8外显子的跳跃增加。结论:U2AF65可以通过表达水平的变化参与调控基因UBQLN1的可变剪接。     

用于前体mRNA剪接机制研究的小基因模型的建立和研究应用

建立可用于选择性前体mRNA剪接分析的小基因模型. 以人或小鼠基因组DNA为模板, 通过PCR扩增获得GluR-B, FGF-2R和Zis小基因片段, 并将其克隆至真核表达载体中, 构建了小基因的质粒. 在此基础上, 将上述3个小基因模型和剪接因子Tra2β1或Zis2表达质粒共转染HeLa细胞, 并用RT-PCR进行了被剪接的小基因产物的半定量检测. 结果表明, 这些小基因可用于细胞水平的基因剪接分析, 利用该技术平台, 发现了Zis2亚型可以促进Zis小基因选择性剪接.     

上皮剪接调节蛋白1的研究进展

上皮剪接调节蛋白1(Epithelial splicing regulatory protein 1,ESRP1)是近年来发现的一种上皮细胞特异性剪接因子,主要通过选择性剪接在转录后水平调节基因的表达,继而影响细胞的功能。研究表明,ESRP1通过调控上皮间质转化、细胞周期进展、氧化还原反应以及脂肪酸代谢等过程,多方面参与肿瘤的发生、发展和对治疗药物的反应。小鼠实验研究表明,ESRP1基因敲除可以导致多种器官发育异常,包括颅面部畸形、皮肤屏障功能受损、肾脏以及耳蜗发育不良等。此外,ESRP1还可以通过调控转录因子的活性以及非编码RNA的生成,提高小鼠成纤维细胞重编程为多能干细胞的效率并维持人胚胎干细胞的多能性。鉴于ESRP1在多个研究领域的重要性,本文对ESRP1常见的下游靶分子、信号通路、以及在生理病理环境下所发挥的功能进行阐述,以期进一步指导基础研究和临床应用。     

拟南芥SDIR1基因转录的选择性剪接

采用PCR及RT-PCR法分别克隆了拟南芥SDIR1基因的DNA和cDNA序列。根据序列比对分析结果,发现了3种不同的转录本,提示SDIR1基因的转录中存在选择性剪接。3种转录本的长度分别为822bp、691bp和666bp,依次命名为：SDIR1-822、SDIR1-691、SDIR1-666。与SDIR1基因的DNA序列及已报道的SDIR1cDNA序列比较,除转录本SDIR1-822包含了完整的编码序列外,其余2种转录本的编码序列都存在不同长度的缺失。其中,SDIR1-691缺失了131bp的片段：第2外显子3′端缺失33bp,第3外显子53bp全部缺失,第4外显子5′端缺失45bp;转录本SDIR1-666缺失了156bp的片段：第3外显子3′端缺失18bp,第4外显子5′端缺失138bp。进而随机挑取101个克隆子对三种转录本的表达比例进行初步分析,结果表明3种分子的比值为SDIR1-822：SDIR1-691：SDIR1-666=26.00：1.33：1.00,反映出SDIR1基因不同转录本在拟南芥中的相对表达量。     

β淀粉样蛋白1-42对BV-2细胞产生一氧化氮功能的刺激作用

目的应用β淀粉样蛋白1-42(β-Amyloid,Aβ1-42)作用于小胶质细胞(microglia,MG),对MG产生一氧化氮(nitric oxide,NO)的作用进行研究.方法应用高度纯化的BV-2小胶质细胞作为体外小胶质细胞模型,测定加入Aβ1-42后细胞上清NO含量及细胞iNOS酶活力;Western blot法测定Aβ对BV-2细胞iNOS蛋白表达的影响,免疫细胞化学方法对iNOS蛋白的表达情况进行观察.结果 Aβ1-42可以刺激BV-2细胞产生NO、提高细胞iNOS酶活性、增加iNOS蛋白质表达,以上作用均具有时间及浓度依赖性.结论 Aβ1-42在体外可通过提高细胞iNOS酶活性、增加iNOS蛋白质表达而增加NO的分泌,为NO发挥神经元毒性作用创造了条件.     

剪接因子SR—蛋白特异的激酶—SRPK1的发现

1977年,当美国麻省理工学院的PhillipSharp和冷泉港实验室的Richard Roberts分别领导的两个研究小组在研究腺病毒(adenov-irus)和其mRNA之间的杂种分子发现了基因割裂(split)现象时,他们立刻感到诺贝尔已带着他的遗产敲开了他们的心扉。果然,一个新的称为RNA剪接Splicing的研究领域从此诞生了,16年后,随着这一领域的不断开拓,当时这一发现的领导者Phillip Sharp和Richard Roberts终于于1993年分享了诺贝尔     

山羊β—酪蛋白基因启动子指导人血清白蛋白基因在小鼠组织中的特异性表达

对本所构建的pcDNA3.1-β6.7hALBm表达载体行尾静脉注射直接转染哺乳期母鼠的活体组织。通过RT－PCR检测hALBm基因在小鼠几种组织中的表达来研究该构建体的组织特异性。结果：构建体在受试鼠组织中的表达显示了较好的乳腺组织特异性。说明构建具有较长5'端上游乳蛋白调控区的乳腺特异性表达载体对组织特异性表达是必需的。     

The splicing regulators Tra and Tra2 are unusually potent activators of pre-mRNA splicing

Sexual differentiation in Drosophila is regulated through alternative splicing of doublesex. Female-specific splicing is activated through the activity of splicing enhancer complexes assembled on multiple repeat elements. Each of these repeats serves as a binding platform for the cooperative assembly of a heterotrimeric complex consisting of the SR proteins Tra, Tra2 and 9G8. Using quantitative kinetic analyses, we demonstrate that each component of the enhancer complex is capable of recruiting the spliceosome. Surprisingly, Tra, Tra2 and 9G8 are much stronger splicing activators than other SR protein family members and their activation potential is significantly higher than expected from their serine/arginine content. 9G8 activates splicing not only through its RS domains but also through its RNA-binding domain. The RS domains of Tra and Tra2 are required but not sufficient for efficient complex assembly. Thus, the regulated assembly of the dsx enhancer complexes leads to the generation of an extended activation domain to guarantee the ‘all or none’ splicing switch that is required during Drosophila sexual differentiation.     

Mutations in tau gene exon 10 associated with FTDP-17 alter the activity of an exonic splicing enhancer to interact with Tra2 beta

Mutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. This exonic enhancer element interacts with human Tra2 beta protein. The interaction between Tra2 beta and the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating splicing. Biochemical studies including in vitro splicing and RNA interference experiments in transfected cells support a role for Tra2 beta protein in regulating alternative splicing of human tau gene. Our results implicate the human tau gene as a target gene for the alternative splicing regulator Tra2 beta, suggesting that Tra2 beta may play a role in aberrant tau exon 10 alternative splicing and in the pathogenesis of tauopathies.     

Protein splicing: selfish genes invade cellular proteins

Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the ‘protein intron’ is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.     

Tra2betal regulates P19 neuronal differentiation and the splicing of FGF-2R and GluR-B minigenes

The present study demonstrates that the expression of Tra2beta1 (Transformer 2-beta1) proteins, an SR (serine/arginine rich) protein, is developmentally up-regulated in a neural-specific pattern. The up-regulation is also observed in RA (retinoic acid) induced neural differentiation of P19 cells. Tra2betal proteins are located in the nuclei of P19 cells, which are consistent with its functional site as an SR protein. The over-expression of Tra2betal proteins promotes RA induced neuronal differentiation of P19 cells. In P19 cells, the splicing of FGF-2R (fibroblast growth factor receptor 2) minigene produces the BEK form, while the alternative splicing of GluR-B (glutamate receptor subunit B) minigene generates two products, the Flop and the Truncated isoforms. Tra2betal inhibits the BEK splicing, but it promotes the Flop splicing. The results therefore suggest that Tra2betal is involved in the regulation of alternative splicing processes during neural development, peculiarly the splicing of FGF-2R and GluR-B genes. Both FGF-2R and GluR-B genes are known to play important roles in neural differentiation.     

Cloning and genetic analysis of tra cistrons of the Tra 2/Tra 3 region of plasmid RP1

Transfer-defective mutants of the 10.4-kb Tra 2/Tra 3 region of RP1 were identified by their ability to be complemented by clones carrying all or part of this region. The respective mutations occurred in six cistrons whose order (traA, B, E, R, P, Q) and location were determined by deletion and insertion mapping. The cistrons occupy a minimum of 5.5 kb with the most distal, traA, spanning the 28.0-kb map position and traR the KpnI site at map position 24.1 kb. Each cistron is expressed independently, as Tn5 or Tn504 insertions in any one cistron do not affect the other five. The phenotypes controlled by each cistron suggest that all contribute to pilus biosynthesis/function while three (traB, R, and P) also contribute to surface exclusion. Given the occurrence of tra cistrons in the "silent" region between Tra 2 and Tra 3 we propose that the epithet "Tra 2" should be used to describe this entire region.     

Regulation of RAGE splicing by hnRNP A1 and Tra2β‐1 and its potential role in AD pathogenesis

The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, full‐length membrane‐bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis, either via interaction of mRAGE with β‐amyloid peptide (Aβ) or inhibition of the mRAGE‐activated signaling pathway. In the present study, we showed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and Transformer2β‐1 (Tra2β‐1) were involved in the alternative splicing of mRAGE and esRAGE. Functionally, two factors had an antagonistic effect on the regulation. Glucose deprivation induced an increased ratio of mRAGE/esRAGE via up‐regulation of hnRNP A1 and down‐regulation of Tra2β‐1. Moreover, the ratios of mRAGE/esRAGE and hnRNP A1/Tra2β‐1 were increased in peripheral blood mononuclear cells from AD patients. The results provide a molecular basis for altered splicing of mRAGE and esRAGE in AD pathogenesis.