Validation of miRNA-mRNA interactions by electrophoretic mobility shift assays.
BACKGROUND
MicroRNAs are small non-coding RNAs involved in gene expression regulation by targeting specific regions in the 3'-UTR of the mRNA of their target genes. This binding leads to a decrease in the protein levels of such genes either by mRNA degradation or mRNA destabilization and translation inhibition. The interaction between a miRNA and its target mRNAs is usually studied by co-transfection of a reporter expression vector containing the 3'-UTR region of the mRNA and an inhibitory or precursor molecule for the miRNA. This approach, however, does not measure the direct and physical interaction between a miRNA and a specific mRNA.


RESULTS
RNA molecules corresponding to miR-224 and to the 3'-UTR of SLC4A4 were incubated together and their interaction studied under different binding conditions using electrophoretic mobility shift assays. A direct and specific interaction between miR-224 and SLC4A4 mRNA was observed. This interaction was abolished in the presence of competitors.


CONCLUSIONS
In this study, we explored a new application for the electrophoretic mobility shift assay and we demonstrated that it is a useful alternative method to assess, in a direct and specific manner, whether a miRNA binds to a specific predicted target mRNA.
Anna
A
Solé
Núria
N
Mencia
Xenia
X
Villalobos
Véronique
V
Noé
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Avenue Diagonal 643, Barcelona E-08028, Spain. cciudad@ub.edu.
Carlos J
CJ
Ciudad
D020413:3' Untranslated Regions / Q000235:genetics*; D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics; D010761:Phosphorus Radioisotopes; D020871:RNA Stability; D012265:Ribonucleotides / Q000737:chemistry* / Q000235:genetics; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000235:genetics
D020413:3' Untranslated Regions; C546140:MIRN224 microRNA, human; D035683:MicroRNAs; D010761:Phosphorus Radioisotopes; D012265:Ribonucleotides; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters