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Updated: May 5, 2026
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Published on: May 30, 2012
MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation
Yvonne Tay1, Jinqiu Zhang, Andrew M Thomson
1Stem Cell and Developmental Biology, Genome Institute of Singapore, Agency for Science Technology and Research, #08-01, Genome, 60 Biopolis Street, Singapore 138672, Singapore.
Abstract:
MicroRNAs (miRNAs) are short RNAs that direct messenger RNA degradation or disrupt mRNA translation in a sequence-dependent manner. For more than a decade, attempts to study the interaction of miRNAs with their targets were confined to the 3' untranslated regions of mRNAs, 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 genes 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.
Insights
MicroRNAs (miRNAs) target coding sequences in genes, not just untranslated regions. This discovery reveals new mechanisms for gene regulation and species-specific miRNA activity.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- MicroRNAs (miRNAs) are key regulators of gene expression.
- Historically, miRNA target sites were assumed to be exclusively in the 3' untranslated regions (UTRs) of messenger RNAs (mRNAs).
- This assumption limited the understanding of miRNA-mediated gene regulation.
Purpose of the Study:
- To investigate the existence and function of miRNA targets within the coding sequences (CDS) of specific genes.
- To explore the role of CDS-located miRNA targets in mouse embryonic stem cell differentiation.
- To challenge the traditional model of miRNA-target interactions.
Main Methods:
- Analysis of mouse Nanog, Oct4, and Sox2 genes for miRNA target sites in their CDS.
- Experimental validation using site-directed mutagenesis to assess the impact of target site disruption.
- Observation of cellular and phenotypic changes following miRNA-mediated targeting of CDS.
Main Results:
- Identification of numerous naturally occurring miRNA target sites within the CDS of Nanog, Oct4, and Sox2.
- Demonstration that specific miRNAs (miR-134, miR-296, miR-470) target these CDS sites during stem cell differentiation.
- Observed transcriptional and morphological changes characteristic of differentiation, with mutations preventing these effects.
Conclusions:
- miRNA targets are not limited to 3' UTRs and are abundant in CDS.
- CDS-located miRNA targets can be species-specific.
- This expands the known mechanisms of miRNA function, supporting an augmented model of gene regulation.
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