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Cell:RNA结合蛋白能抑制miRNAs作用

来源:生物谷 2007-12-29 00:14

生物谷报道:来自荷兰癌症研究院(The Netherlands Cancer Institute)肿瘤生物学部,德国马克斯·普朗克生物物理化学研究院(Max-Planck-Institute for Biophysical Chemistry),丹麦哥本哈根大学等多国研究人员组成的研究团队发现了一种进化上保守的RNA结合蛋白能抑制几种miRNAs与其靶标mRNA位点相互作用,阻碍miRNA的功能。这不仅揭示了这种在保护某些mRNAs免受miRNA介导的抑制中的新作用,也揭示了miRNA调控中的一条新途径。这一研究成果公布在最新一期的Cell杂志上。

微小RNA (microRNA,简称miRNA)是生物体内源长度约为20-23个核苷酸的非编码小RNA,通过与靶mRNA的互补配对而在转录后水平上对基因的表达进行负调控,导致mRNA的降解或翻译抑制。到目前为止,已报道有几千种miRNA存在于动物、植物、真菌等多细胞真核生物中,进化上高度保守。

在哺乳动物中,miRNAs的基因抑制作用是通过其一段长为6-8nt的核苷酸与mRNAs3’端非翻译区域相结合实现的,让研究人员感兴趣的是,不仅是miRNA靶标位点,而且在其邻近位置的序列也是进化上高度保守的。

因此在这篇文章中,研究人员假定mRNAs中保守区域也许具有作为miRNA活性调节的一个导入平台的功能,为了证明这一点,他们针对一个进化上保守的RNA结合蛋白(RNA-binding protein,RBP,生物谷注):Dnd1进行了研究。通过实验研究人员发现这种蛋白可以通过结合在mRNAs上,抑制miRNAs与其靶标位点相互作用,来阻碍人类细胞和斑马鱼中原代生殖细胞中几种miRNAs的功能。

进一步研究发现这种Dnd1的作用是通过miRNA靶定的mRNAs上尿嘧啶富集区域介导的,因此这一研究结果揭示了Dnd1在保护某些mRNAs免受miRNA介导的抑制中的新作用,也揭示了miRNA调控中的一条新途径。

2007年Science十大科学进展以icroRNA、人工制造的微生物、新的计算机芯片材料等作为了2008年应该注意的领域,其中小分子RNA这个让人目眩神迷的“神奇小子”可以说发展前景不可限量。

然而无论从基础研究还是治疗药物学研究方面,小分子RNA研究还属于幼年期。2007年研究人员发现miRNAs这种非编码RNAs不仅只用于抑制基因表达,而且也具有转录激活这一完全相反的作用,同时在miRNA基础功能研究方面,研究人员还发现miRNA能引起肿瘤扩散,促进实体肿瘤转移,侵染其它组织,这些都说明对于miRNA的功能了解我们还尚未看清楚其“庐山真面目”。

在这一年里,研究人员也寻找到了miRNAs这一家族里的新成员,譬如金海玲等人发现long short interfering RNAs (lsiRNAs),林海帆等人确定了piRNA在基因中所起的关键作用等等,这

些都说明小RNA家族和小RNA介导的基因调控远比之前预想的复杂。不知在2008年我们是否能看到更多的小RNA新家族呢?

而在RNAi技术方面,关键的传递方法07年也获得了一些进展:研究人员成功阐明了哺乳动物中与脂肪酸结合的siRNA如何被吸收的机制,这对于siRNA递送来说意义重大,近期来自国内的研究人员成功解决了把RNA干扰药物特异性导入体内细胞的难题——他们通过病毒载体,把人工合成的蛋白精确导入癌干细胞中,恢复了let-7的表达。相信在2008年将会有更多新鲜的miRNA成果纷纷呈递。

生物谷推荐英文原文:

Cell, Vol 131, 1273-1286, 28 December 2007

Article

RNA-Binding Protein Dnd1 Inhibits MicroRNA Access to Target mRNA

Martijn Kedde,1 Markus J. Strasser,2 Bijan Boldajipour,2 Joachim A.F. Oude Vrielink,1 Krasimir Slanchev,2,5 Carlos le Sage,1 Remco Nagel,1 P. Mathijs Voorhoeve,1 Josyanne van Duijse,1 Ulf Andersson Ørom,3 Anders H. Lund,3 Anastassis Perrakis,4 Erez Raz,2, and Reuven Agami1,

1 The Netherlands Cancer Institute, Division of Tumor Biology, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands
2 Max-Planck-Institute for Biophysical Chemistry, Germ Cell Development, Am Fassberg 11, 37070 Goettingen, and Institute for Cell Biology, ZMBE, Center for Molecular Biology of Inflammation, University of Münster, Münster 48149, Germany
3 Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, 2200N, Copenhagen, Denmark
4 The Netherlands Cancer Institute, Division of Molecular Carcinogenesis, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands

Corresponding author
Erez Raz
erezraz@uni-muenster.de

Corresponding author
Reuven Agami
r.agami@nki.nl

MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6–8 nucleotides (nt) to associate with 3′ untranslated regions (3′UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus, our data unravel a novel role of Dnd1 in protecting certain mRNAs from miRNA-mediated repression.

  Figure 1. Dnd1 Counteracts the Inhibition of p27 Expression by miR-221

(A) Conservation analysis of p27-3′UTR from human to fish (from Kent et al. [2002]). The positions of the two target sequences of miR-221 are marked.

(B) Expression vectors for miR-221 and human Dnd1 (huDnd1) were cotransfected with the indicated luciferase constructs. Relative luciferase activity is the ratio between firefly luciferase and renilla control luciferase, adjusted to 100%. An immunostaining with anti-HA antibody demonstrates the expression of huDnd1 while H2B-GFP was used to control transfection efficiency. The results are represented as means and SD from three independent experiments.

(C) HEK293T cells were transfected with the indicated constructs and whole-cell lysates were immunostained with anti-Tubulin, p27, and HA antibodies. p27 protein level was analyzed using Tina 2.0 software (Raytest, Sheffield, UK).

(D and E) Similar to (B), only that several RBPs, as well as the zebrafish Dnd1 homolog (drDnd1) and a mutant in the RNA-binding domain (drDnd1Y104C), were cotransfected together with pGL3-p27-3′UTR and renilla luciferase control.

(F) HEK293T cells were transfected with the indicated constructs and subjected to RPA with probes to detect p27 mRNA and control cyclophilin and to immunoblot analysis using p27 and control Tubulin antibodies. Quantification of protein levels was performed using Tina 2.0 software (Raytest; Sheffield, UK).

(G) Tera1 cells were transfected with shDnd1 and subjected to quantitative RT-PCR analysis for LATS2, Dnd1, and GAPDH control. The results are represented as means and SD from three independent experiments.

(H) Similar to (B), Tera1 cells were transfected with the indicated constructs.

微小RNA (microRNA,简称miRNA)是生物体内源长度约为20-23个核苷酸的非编码小RNA,通过与靶mRNA的互补配对而在转录后水平上对基因的表达进行负调控,导致mRNA的降解或翻译抑制。到目前为止,已报道有几千种miRNA存在于动物、植物、真菌等多细胞真核生物中,进化上高度保守。在植物和动物中,miRNA虽然都是通过与其靶基因的相互作用来调节基因表达,进而调控生物体的生长发育,但miRNA执行这种调控作用的机理却不尽相同。

1993年,首次在秀丽隐杆线虫(Caenorhabditiselegans)中发现microRNAs,现已证实,miRNA 广泛存在于真核生物细胞内,是最大的基因家族之一,大约占到整个基因组的1%,在精细调控基因表达及生物生长发育过程方面发挥着重要作用。任何miRNAs的失调都会导致细胞调控事件的剧变。最近研究表明,miRNA在生物体内的多样化调控途径中扮演着关键性角色,包括控制发育进程、细胞分化、细胞凋亡、细胞分裂以及器官的发育。miRNA与其靶分子组成了一个复杂的调控网络,如某一特定的miRNA 可以与多个mRNA 分子结合而发挥调控功能,反之,不同的miRNA 分子也可以结合在同一mRNA 分子上,协同调控此mRNA 分子的表达。

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