MicroRNAs (miRNAs) are short RNAs that direct messenger RNA degradation or disrupt mRNA translation in a sequence-dependent manner1,2,3,4,5,6,7. For more than a decade, attempts to study the interaction of miRNAs with their targets were confined to the 3′ untranslated regions of mRNAs1, fuelling an underlying assumption that these regions are the principal recipients of miRNA activity. Here we focus on the mouse Nanog, Oct4 (also known as Pou5f1) and Sox2 genes8,9,10,11 and demonstrate the existence of many naturally occurring miRNA targets in their amino acid coding sequence (CDS). Some of the mouse targets analysed do not contain the miRNA seed, whereas others span exon–exon junctions or are not conserved in the human and rhesus genomes. miR-134, miR-296 and miR-470, upregulated on retinoic-acid-induced differentiation of mouse embryonic stem cells, target the CDS of each transcription factor in various combinations, leading to transcriptional and morphological changes characteristic of differentiating mouse embryonic stem cells, and resulting in a new phenotype. Silent mutations at the predicted targets abolish miRNA activity, prevent the downregulation of the corresponding genes and delay the induced phenotype. Our findings demonstrate the abundance of CDS-located miRNA targets, some of which can be species-specific, and support an augmented model whereby animal miRNAs exercise their control on mRNAs through targets that can reside beyond the 3′ untranslated region.
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We thank T. Huynh for assistance with building the rna22 pipeline that was used to make the miRNA target predictions, and H. Zhang and Y. L. Lee for their help with some of the constructs. The work of Y.T., J.Z., A.M.T. and B.L. was supported by the Agency for Science, Technology and Research of Singapore. Y.T. was the recipient of an A*STAR graduate scholarship. B.L. was also partially supported by National Institutes of Health grants DK47636 and AI54973.
Author Contributions Y.T. carried out the experiments and assisted with writing the paper. J.Z. assisted with the experiments. A.M.T. assisted with and supervised the experiments, and assisted with writing the paper. B.L. supervised the experiments and supported the experimental aspects of the research. I.R. designed the research and experiments, carried out the computational analyses and wrote the paper.
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Tay, Y., Zhang, J., Thomson, A. et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 455, 1124–1128 (2008). https://doi.org/10.1038/nature07299
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