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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。
