A Microarray-based Approach Identifies ADP Ribosylation Factor-like Protein 2 as a Target of microRNA-16

Yanye Dong,Jiangning Chen,Jigang Guo,Yuan Yin,Kehui Wang,Chen-Yu Zhang,Xi Chen,Peng Li,Yujing Zhang,Jing Li,Ke Zen,Hongwei Liang,Dongxia Hou,Xing Cai,Junfeng Zhang,Bowen Yu

doi:10.1074/jbc.m110.178335

Abstract

microRNAs (miRNAs) are generally thought to negatively regulate the expression of their target genes by mRNA degradation or by translation repression. Here we show an efficient way to identify miRNA target genes by screening alterations in global mRNA levels following changes in miRNA levels. In this study, we used mRNA microarrays to measure global mRNA expression in three cell lines with increased or decreased levels of miR-16 and performed bioinformatics analysis based on multiple target prediction algorithms. For further investigation among the predicted miR-16 target genes, we selected genes that show an expression pattern opposite to that of miR-16. One of the candidate target genes that may interact with miR-16, ADP-ribosylation factor-like protein 2 (ARL2), was further investigated. First, ARL2 was deduced to be an ideal miR-16 target by computational predictions. Second, ARL2 mRNA and protein levels were significantly abolished by treatment with miR-16 precursors, whereas a miR-16 inhibitor increased ARL2 mRNA and protein levels. Third, a luciferase reporter assay confirmed that miR-16 directly recognizes the 3'-untranslated region (3'-UTR) of ARL2. Finally, we showed that miR-16 could regulate proliferation and induce a significant G0/G1 cell cycle arrest, which was due at least in part, to the down-regulation of ARL2. In summary, the present study suggests that integrating global mRNA profiling and bioinformatics tools may provide the basis for further investigation of the potential targets of a given miRNA. These results also illustrate a novel function of miR-16 targeting ARL2 in modulating proliferation and cell cycle progression.

Highlights

K-Ras4B modulates downstream signaling at different lipid microdomains
The non-farnesylated peptide binds all phospholipid nanodiscs with increasing KD values: 1.59 Ϯ 0.01, 2.10 Ϯ 0.01, and 5.06 Ϯ 0.04 ␮M for DPPC, DOPC, and DOPS nanodiscs, respectively
A more disordered packing would allow for insertion of the farnesyl groups into lipid bilayers, thereby decreasing cooperativity

Summary

Background

K-Ras4B modulates downstream signaling at different lipid microdomains. Results: K-Ras4B farnesyl group spontaneously inserts into the disordered lipid microdomains, but phosphorylation prohibits the farnesyl membrane insertion. HVR farnesylation plays an important role in Ras signaling [14], but the structural mechanisms and consequences of farnesyl-induced membrane binding are poorly understood. Activated K-Ras (G12V) interacts with non-raft regions [22], suggesting that K-Ras4B is preferentially localized in liquid-disordered lipid domains [23]. Studies of the modeled HVR peptides verify that the farnesyl group spontaneously inserts into the lipid bilayer containing unsaturated phospholipids, but the insertion is restricted by phosphorylation. We speculate that if the farnesyl group is not inserted into the membrane, it may serve in cooperative binding to other K-Ras4B molecules, leading to dimerization In this case, the protein binds the membrane through electrostatic interactions between its positively charged HVR and the negatively charged membrane phospholipids. In the case of disordered phospholipid domains, the farnesyl group burial in the phospholipids tends to hinder its interactions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION