生物谷報道：來自荷蘭癌癥研究院（The Netherlands Cancer Institute）腫瘤生物學部,，德國馬克斯·普朗克生物物理化學研究院（Max-Planck-Institute for Biophysical Chemistry），丹麥哥本哈根大學等多國研究人員組成的研究團隊發(fā)現(xiàn)了一種進化上保守的RNA結(jié)合蛋白能抑制幾種miRNAs與其靶標mRNA位點相互作用,，阻礙miRNA的功能。這不僅揭示了這種在保護某些mRNAs免受miRNA介導的抑制中的新作用,，也揭示了miRNA調(diào)控中的一條新途徑,。這一研究成果公布在最新一期的Cell雜志上。
微小RNA (microRNA,，簡稱miRNA)是生物體內(nèi)源長度約為20－23個核苷酸的非編碼小RNA,，通過與靶mRNA的互補配對而在轉(zhuǎn)錄后水平上對基因的表達進行負調(diào)控，導致mRNA的降解或翻譯抑制,。到目前為止,，已報道有幾千種miRNA存在于動物、植物,、真菌等多細胞真核生物中,，進化上高度保守。
在哺乳動物中,，miRNAs的基因抑制作用是通過其一段長為6－8nt的核苷酸與mRNAs3’端非翻譯區(qū)域相結(jié)合實現(xiàn)的,，讓研究人員感興趣的是，不僅是miRNA靶標位點,，而且在其鄰近位置的序列也是進化上高度保守的,。
因此在這篇文章中，研究人員假定mRNAs中保守區(qū)域也許具有作為miRNA活性調(diào)節(jié)的一個導入平臺的功能,，為了證明這一點,，他們針對一個進化上保守的RNA結(jié)合蛋白（RNA-binding protein，RBP,，生物谷注）：Dnd1進行了研究,。通過實驗研究人員發(fā)現(xiàn)這種蛋白可以通過結(jié)合在mRNAs上，抑制miRNAs與其靶標位點相互作用,，來阻礙人類細胞和斑馬魚中原代生殖細胞中幾種miRNAs的功能,。
進一步研究發(fā)現(xiàn)這種Dnd1的作用是通過miRNA靶定的mRNAs上尿嘧啶富集區(qū)域介導的，因此這一研究結(jié)果揭示了Dnd1在保護某些mRNAs免受miRNA介導的抑制中的新作用,，也揭示了miRNA調(diào)控中的一條新途徑,。
2007年Science十大科學進展以icroRNA、人工制造的微生物,、新的計算機芯片材料等作為了2008年應該注意的領(lǐng)域,，其中小分子RNA這個讓人目眩神迷的“神奇小子”可以說發(fā)展前景不可限量。
然而無論從基礎(chǔ)研究還是治療藥物學研究方面,，小分子RNA研究還屬于幼年期,。2007年研究人員發(fā)現(xiàn)miRNAs這種非編碼RNAs不僅只用于抑制基因表達,，而且也具有轉(zhuǎn)錄激活這一完全相反的作用，同時在miRNA基礎(chǔ)功能研究方面,，研究人員還發(fā)現(xiàn)miRNA能引起腫瘤擴散,，促進實體腫瘤轉(zhuǎn)移，侵染其它組織,，這些都說明對于miRNA的功能了解我們還尚未看清楚其“廬山真面目”,。
在這一年里，研究人員也尋找到了miRNAs這一家族里的新成員,，譬如金海玲等人發(fā)現(xiàn)long short interfering RNAs （lsiRNAs）,，林海帆等人確定了piRNA在基因中所起的關(guān)鍵作用等等，這
些都說明小RNA家族和小RNA介導的基因調(diào)控遠比之前預想的復雜,。不知在2008年我們是否能看到更多的小RNA新家族呢,？
而在RNAi技術(shù)方面，關(guān)鍵的傳遞方法07年也獲得了一些進展：研究人員成功闡明了哺乳動物中與脂肪酸結(jié)合的siRNA如何被吸收的機制,，這對于siRNA遞送來說意義重大,，近期來自國內(nèi)的研究人員成功解決了把RNA干擾藥物特異性導入體內(nèi)細胞的難題——他們通過病毒載體，把人工合成的蛋白精確導入癌干細胞中,，恢復了let-7的表達,。相信在2008年將會有更多新鮮的miRNA成果紛紛呈遞。
生物谷推薦英文原文：
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
[email protected]
Corresponding author
Reuven Agami
[email protected]
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,，簡稱miRNA)是生物體內(nèi)源長度約為20－23個核苷酸的非編碼小RNA,，通過與靶mRNA的互補配對而在轉(zhuǎn)錄后水平上對基因的表達進行負調(diào)控，導致mRNA的降解或翻譯抑制,。到目前為止,，已報道有幾千種miRNA存在于動物、植物,、真菌等多細胞真核生物中,，進化上高度保守。在植物和動物中,，miRNA雖然都是通過與其靶基因的相互作用來調(diào)節(jié)基因表達,，進而調(diào)控生物體的生長發(fā)育，但miRNA執(zhí)行這種調(diào)控作用的機理卻不盡相同,。
1993年,，首次在秀麗隱桿線蟲(Caenorhabditiselegans)中發(fā)現(xiàn)microRNAs，現(xiàn)已證實,，miRNA 廣泛存在于真核生物細胞內(nèi),，是最大的基因家族之一，大約占到整個基因組的1%,，在精細調(diào)控基因表達及生物生長發(fā)育過程方面發(fā)揮著重要作用,。任何miRNAs的失調(diào)都會導致細胞調(diào)控事件的劇變,。最近研究表明，miRNA在生物體內(nèi)的多樣化調(diào)控途徑中扮演著關(guān)鍵性角色,，包括控制發(fā)育進程、細胞分化,、細胞凋亡,、細胞分裂以及器官的發(fā)育。miRNA與其靶分子組成了一個復雜的調(diào)控網(wǎng)絡(luò),，如某一特定的miRNA 可以與多個mRNA 分子結(jié)合而發(fā)揮調(diào)控功能,，反之，不同的miRNA 分子也可以結(jié)合在同一mRNA 分子上,，協(xié)同調(diào)控此mRNA 分子的表達,。
