Global and precise identification of functional miRNA targets in mESCs by integrative analysis

Abstract

MicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Previous works suggest that up to 60% of mammalian transcripts might be subject to miRNA-mediated regulation, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrate transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles. Using this integrative approach, combined with molecular validation data, we present evidence that < 10% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identify TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions.

Analysis of a mouse germ cell tumor model establishes pluripotency-associated miRNAs as conserved serum biomarkers for germ cell cancer detection

“…( B ) Significantly enriched (FDR < 0.05) mirDB gene sets associated with the miR-290-295 cluster in TR-differentiated cells compared to parental EC cell lines with normalized enrichment scores (NES) as determined by GSEA. ( C ) Volcano plots showing differential gene expression of the 106 candidate target genes of the miR-290-295 cluster from Schaefer et al 40 Gene names of experimentally determined direct targets of the miR-290-295 cluster are labelled. Genes that were significantly upregulated in TR-differentiated cells are in red, genes that were significantly downregulated in TR-differentiated cells are in blue, and genes that were not significantly differentially expressed are in grey.…”

“…Data were visualized using GraphPad Prism. 40 Gene names of experimentally determined direct targets of the miR-290-295 cluster are labelled. Genes that were significantly upregulated in TR-differentiated cells are in red, genes that were significantly downregulated in TR-differentiated cells are in blue, and genes that were not significantly differentially expressed are in grey.…”

“…In total, 26 target genes were identified from 16 publications, which were focused primarily on ES cells (Supplemental Table 1). [22][23][24][32][33][34][35][36][37][38][39][40][41][42][43][44] Of these 26 genes, 13 were significantly upregulated and five were significantly downregulated in TR3 cells compared to EC3 cells (Supplemental Figure 2). Similarly, 14 were significantly upregulated and five were significantly downregulated in TR11 cells compared to EC11 cells.…”

“…23 Schaefer et al, recently identified 360 genes predicted to be targets of the mouse miR-290-295 cluster. 40 The target genes were identified through an integrative analysis of multiple datasets including computationally-predicted miR-290-295 targets in TargetScan, AGO2-miRNA binding and loading data from ES cells, and genes upregulated in global miRNA KO ES cells. 106 of these 360 candidate target genes were found to be upregulated following miR-290-295 KO in ES cells.…”

Selecting Normalizers for MicroRNA RT-qPCR Expression Analysis in Murine Preimplantation Embryos and the Associated Conditioned Culture Media

“…This widespread regulation has spurred their popularity, driven further by their potential use as diagnostic biomarkers owing to their considerable variety and extracellular stability [ 3 ]. miRNAs are expressed within a wide range of plants and animals, including mammalian preimplantation embryos [ 4 , 5 ] wherein miRNAs regulate developmentally critical features such as inner cell mass pluripotency and proliferation [ 6 , 7 ]. Extracellular miRNAs found within the surrounding spent culture media are also finding application as accessible biomarkers indicative of the developmental state [ 8 ].…”