Lytle J Robin, Yario Therese A, Steitz Joan A
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536, USA.
Proc Natl Acad Sci U S A. 2007 Jun 5;104(23):9667-72. doi: 10.1073/pnas.0703820104. Epub 2007 May 29.
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.
在动物中，微小RNA（miRNA）与靶标mRNA的3'非翻译区（UTR）结合并干扰翻译，尽管抑制蛋白质合成的确切机制仍不清楚。内源性mRNA的编码区或5'UTR中功能性miRNA结合位点尚未被鉴定。我们研究了将miRNA靶位点引入含有内部核糖体进入位点（IRES）的荧光素酶报告基因mRNA的5'UTR的效果，以便微小核糖核蛋白复合体的潜在空间位阻不会干扰翻译起始。在表达内源性let-7a miRNA的人HeLa细胞中，这些含有来自秀丽隐杆线虫lin-41 3'UTR的5' let-7互补位点的含IRES报告基因的翻译效率受到抑制。同样，当人AGO2与5'或3'UTR相连时，含IRES的报告基因的翻译也受到抑制。有趣的是，DNA转染方法影响了我们观察miRNA介导的抑制作用的能力。我们的结果表明，与靶标mRNA上的任何位置结合在机制上足以使微小核糖核蛋白在起始下游的某个步骤发挥翻译抑制作用。
