title: Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5' UTR as in the 3' UTR.
abstract: In animals, microRNAs (miRNAs) bind to the 3' UTRs of their target mRNAs and interfere with translation, although the exact mechanism of inhibition of protein synthesis remains unclear. Functional miRNA-binding sites in the coding regions or 5' UTRs of endogenous mRNAs have not been identified. We studied the effect of introducing miRNA target sites into the 5' UTR of luciferase reporter mRNAs containing internal ribosome entry sites (IRESs), so that potential steric hindrance by a microribonucleoprotein complex would not interfere with the initiation of translation. In human HeLa cells, which express endogenous let-7a miRNA, the translational efficiency of these IRES-containing reporters with 5' let-7 complementary sites from the Caenorhabditis elegans lin-41 3' UTR was repressed. Similarly, the IRES-containing reporters were translationally repressed when human Ago2 was tethered to either the 5' or 3' UTR. Interestingly, the method of DNA transfection affected our ability to observe miRNA-mediated repression. Our results suggest that association with any position on a target mRNA is mechanistically sufficient for a microribonucleoprotein to exert repression of translation at some step downstream of initiation.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536, USA.
authors.forename: J Robin
authors.initials: JR
authors.lastname: Lytle
authors.forename: Therese A
authors.initials: TA
authors.lastname: Yario
authors.forename: Joan A
authors.initials: JA
authors.lastname: Steitz
mesh_terms: D020121:5' Untranslated Regions / Q000378:metabolism*; D000818:Animals; D060565:Argonaute Proteins; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000235:genetics; D012261:Ribonucleoproteins / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D020121:5' Untranslated Regions; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D029742:Caenorhabditis elegans Proteins; D015852:Eukaryotic Initiation Factor-2; C410586:LIN-41 protein, C elegans; D035683:MicroRNAs; D012261:Ribonucleoproteins; D014157:Transcription Factors; C487931:mirnlet7 microRNA, human; D008156:Luciferases