Post-transcriptional regulation of pluripotency by microRNAs and RNA binding proteins
Early mammalian development is driven by precise molecular changes that enable cells to establish, maintain, and exit from the pluripotent state, which is characterized by ability to generate all of the cells that comprise the adult mammal. Embryonic stem cells (ESCs) can be used to model the developmental progression through which pluripotent cells become differentiated somatic cells, and remarkably, ESC-like cells called induced pluripotent stem cells (iPSCs) can be generated from somatic cells like fibroblasts through a process called reprogramming or de-differentiation. Differentiation and de-differentiation are both highly regulated by post-transcriptional mechanisms mediated in part by non-coding RNAs and RNA binding proteins (RBPs). Here, we investigated the roles of microRNAs (miRNAs) and RBPs in the regulation of pluripotency.
Using a fluorescent reporter system, we found that the miR-290 and miR-302 miRNA families are expressed in that order during embryonic development and ESC differentiation but that the loci are transcriptionally activated in a stochastic manner during reprogramming. Addition of the reprogramming enhancer Sall4, however, promoted the transcription of the miR-302 locus before that of miR-290, suggesting that reprogramming does not necessarily represent development in reverse and that the sequence of regulatory changes that occur during the process depends on the specific cocktail of reprogramming factors used. Surprisingly, we found that, despite their expression in pluripotent cells, miR-290 and miR-302 are also both dispensable for the generation of iPSCs.
In a separate set of studies investigating the role of RBPs in pluripotency, we performed an RNAi screen in an ESC differentiation system that allowed us to uncover NF45 and NF90/NF110 as factors important for maintaining pluripotency. Our molecular, cellular, and genomic analyses of NF45 and NF90/NF110 knockdown and knockout ESCs suggested that NF45 and NF90/NF110 regulate the expression of one another and function both independently and in complexes to promote ESC proliferation and proper differentiation.
The studies described here demonstrate the importance and complexity of miRNA and RBP control over the pluripotent state. Developing a more nuanced understanding of these post-transcriptional regulatory processes will enable the more effective and informed use of pluripotent cells to address clinical problems in the future.