Pluripotency-Associated microRNAs in Early Vertebrate Embryos and Stem Cells

Abstract

MicroRNAs (miRNAs), small non-coding RNA molecules, regulate a wide range of critical biological processes, such as proliferation, cell cycle progression, differentiation, survival, and apoptosis, in many cell types. The regulatory functions of miRNAs in embryogenesis and stem cell properties have been extensively investigated since the early years of miRNA discovery. In this review, we will compare and discuss the impact of stem-cell-specific miRNA clusters on the maintenance and regulation of early embryonic development, pluripotency, and self-renewal of embryonic stem cells, particularly in vertebrates.

Keywords: cell cycle regulators; embryonic development; embryonic stem cells; microRNA clusters.

Figure 1

Genome organization and multiple sequence alignment of miR-302/367 precursor coding regions in humans, mice, rabbits, and chickens. (a) Humans, mice, rabbits, and chikens exhibit a common cluster structure, comprising four miR-302s and one miR-367. Interestingly, chikens express an extra miRNA, gga-miR-1811, within the miR-302/367 cluster. (b) Multiple sequence alignment of miR-302/367 precursor coding regions in humans, mice, rabbits, and chickens represent high similarity of this cluster. The seed sequences are highlighted in yellow; mismatches are shown in red. Mature -5p and -3p miRNA sequences are shown in blue. Mature sequence of gga-miR-1811 is shown in green. The asterisks indicate sequence homology.

Figure 2

Main targets of miR-302/367 cluster. The miR-302-367 cluster plays a central role in maintaining ESC pluripotency and self-renewal by regulating different signaling pathways. The cytoplasmic mature miR-302/367 gene products positively regulate self-renewal by modulating G1/S transition through direct targeting of the cell cycle regulators, such as CYCLIN D1/D2 and CDK4/6. Additionally, they promote S phase entry via an alternative Rb-independent pathway. These mature miRNA products also maintain the pluripotency of ESCs by targeting Nodal/Activin inhibitors, such as LEFTY. They also promote BMP signaling through direct targeting of BMP inhibitors (TOB2, DAZAP2, and SLAIN1), thus maintain pluripotency by repressing neural differentiation. Moreover, miR-302/367 members promote reprogramming and iPS cell establishment through induction of global DNA demethylation by post-transcriptional suppression of epigenetic regulators such as AOF1/2, MECP1/2, and MBD2.

Figure 3

Genome organization and multiple sequence alignment of human C19MC cluster and three rabbit homologs. (a) Three rabbit homologues pre-miRNAs of human C19MC including ocu-miR-512, ocu-miR-498, and ocu-miR-520e are located on contig ChrUn0226 in same order as human C19MC on Chr.19. (b) Multiple sequence alignment represented high sequence similarity of rabbit mature miRNAs with their human homologues. Ocu-miR-520e shares the common consensus seed sequence of “AAGTGCT” with its human homologs, but ocu-miR-512 harbors a slightly different seed sequence. The seed sequences are highlighted in yellow. Mismatches in seed sequences are shown in red. The asterisks indicate sequence homology.

Figure 4

Genome organization and multiple sequence alignment of mmu-miR-290/295, ocu-miR-290/295, and hsa-miR-371/373 precursors in mice, humans, and rabbits. (a) miR-371/373, a human ortholog of the mmu-miR-290/295 cluster, is located in close proximity to C19MC on chromosome 19 and comprises of four pre-miRNAs. Rabbit homologs of the mmu-miR-290/295 cluster are located on the reverse strand of a short pseudo-chromosome (chrUn0226) and are comprised of three mature miRNAs. Similar to hsa-miR-371/373, the ocu-miR290/295 cluster is located 3′ to the rabbit C19MC. (b) All pre-miRNAs of mmu-miR-290/295 and hsa-miR-371/373 share ESC-specific seed sequence of “AAGTGC” except for mmu- miR-293 pre-miRNA. From rabbit cluster only ocu-miR-294 contains the same seed sequence. The seed sequences are highlighted in yellow. Mismatches in seed sequences are shown in red. Mature -5p and -3p miRNA sequences are shown in blue. The asterisks indicate sequence homology.

Figure 5

Main targets of miR-290/294 cluster. The miR-290/295 cluster modulates both stemness and differentiation characteristics of mouse ESCs and embryos by regulating cell cycle, de novo DNA methylation, apoptosis, and transcription of pluripotent-associated transcription factors. The cytoplasmic mature miR-290/295 members maintain pluripotency of mESC by accelerating G1 and extending S phase through direct targeting of cell cycle inhibitors such as p21, Lats2, Wee1, and Fbxl5, and also repressing S/G2 transition. These mature miRNAs also incorporate Prc2, Ezh2 and Suz12 into promoters of bivalent differentiation genes. The miR-290/295 cluster together with miR-302/376 promote transition from naïve to primed pluripotency by enhancing the activity of MEK pathway through direct repression of Akt1. These mature miRNAs partially favor pluripotency upon differentiation by direct targeting of Dkk-1, a Wnt pathway inhibitor, resulting in upregulation of c-Myc. The mature miR-290/295 gene products also modulate DNA methylation in the pre-implantation embryos by targeting of Rbl2 and subsequent inhibition of Dnmt3b. The miR-290/295 cluster also enhances the reprogramming of somatic cells and the quality of mouse iPSCs through post-transcriptional suppression of Ash1l expression and consequently downregulation of HOX genes, which in turn promote reprogramming through polycomb-mediated gene silencing.