RNA-based regulation of pluripotency Jane E. Wright and Rafal Ciosk Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland Pluripotent cells have the unique ability to differentiate into diverse cell types. Over the past decade our under- standing of the mechanisms underlying pluripotency, and particularly the role of transcriptional regulation, has in- creased dramatically. However, there is growing evidence for ‘RNA-based’ regulation of pluripotency. We use this term to describe control of gene expression by RNA- binding proteins (RBPs) and regulatory non-coding RNAs (ncRNAs). These molecules bind to specific elements within mRNAs and, by recruiting various effectors, affect many aspects of mRNA regulation. Here, we discuss the role of RBPs and ncRNAs in both the induction and main- tenance of pluripotency. We highlight and contrast exam- ples from pluripotent cell lines and in vivo systems while discussing the connection to transcriptional regulators. Pluripotency is controlled at every level of gene expression Pluripotency describes the potential of a cell to differenti- ate into cells of each of the three embryonic germ layers. In development, pluripotency is restricted to cells of an early embryo and, in regenerating organisms such as planaria, to adult pluripotent stem cells. In addition, pluripotent cell lines, derived primarily from germ cells or early embryos, can be grown ex vivo, for example as embryonic stem (ES), germ (EG), or carcinoma (EC) cells (Figure 1; see Glossary). Finally, so-called induced pluripotent stem (iPS) cells can be produced from non-pluripotent cells by artificially ‘reprogramming’ them. In recent years tremendous prog- ress has been made towards understanding the molecular mechanisms underlying pluripotency. This is particularly true for transcriptional regulation mediated by ‘pluripo- tency transcription factors’ (TFs) such as OCT4 (also known as POU5F1), SOX2, and NANOG. There are many excellent reviews describing the control of pluripotency at a transcriptional level (e.g., [1]). By contrast, the main focus of this review is the ‘RNA-based’ regulation of pluripotency that is mediated by ncRNAs and RBPs, which mostly function post-transcriptionally. Among the ncRNAs, we mostly focus on microRNAs (miRNAs) because they have been connected to pluripotency most convincingly. For the same reason we have included long non-coding RNAs (lncRNAs) even though in this context they appear to function predominantly at the transcriptional level. RNA-based regulation is prominent in germ cells and early embryos and is critical to their development. Because pluripotent stem cells are derived from early embryos or germ cells with relative ease, RNA-based regulation might be expected to be similarly important for their formation and maintenance. All steps of the mRNA life-cycle can be regulated and thus influence protein synthesis. However, it is the cytoplasmic regulation of mature mRNAs that appears to be the most common in development. The mRNA target specificity is mediated via diverse RNA elements typically found in the untranslated regions (UTRs), which bind RBPs and miRNAs. These, in turn, recruit diverse ‘effectors’ that can regulate mRNA expres- sion by, for example, affecting its polyadenylation, capping, or association with translation factors (Figure 2). All of these mechanisms ultimately determine how efficiently an mRNA is translated into protein. There are several possi- ble advantages of using regulation at an mRNA level to control pluripotency. In cells (such as oocytes) that express mRNAs potentially supporting alternative cell fates, mRNA-level regulation allows selective expression of mRNAs promoting a specific differentiation program. Review Glossary Blastocyst: a developmental stage in a pre-implantation mammalian embryo. A blastocyst consists of an inner cell mass (ICM), which later forms the embryo, and an outer layer of cells, the trophoblast, which forms the placenta. Embryonic stem cells (ESCs): pluripotent stem cells that are derived from the inner cell mass of the blastocyst. These cells are capable of dividing in culture without differentiating. Epiblast: the inner layer of the developing embryo that originates from the ICM. It establishes the body plan of the developing embryo after implantation. Cells that make up the epiblast are pluripotent. Epiblast stem cells (EpiSC): pluripotent cells that are derived from the post- implantation mouse epiblast. Induced pluripotent stem cells (iPSCs): pluripotent cells that are typically derived from somatic differentiated cells by cellular ‘reprogramming’, which can be induced by various exogenous factors. Long non coding RNA (lncRNA): a non-coding RNA longer than 200 nucleotides with little or no protein coding potential. Long intergenic non coding RNA (lincRNA): a class of lncRNAs that is transcribed from an intergenic region of the genome. Oocyte to embryo (or maternal to zygotic) transition: the transition from an egg to an early embryo that is governed by maternally provided factors. After this transition, the embryo relies on its own factors for its development. MicroRNAs (miRNAs): a class of naturally occurring, small non-coding RNA molecules, 21–24 nucleotides in length. They are partially complementary to one or more mRNAs, and their binding to mRNA, together with Argonaute proteins, typically results in translational repression and/or degradation of mRNA. Multipotent adult germline stem cells (maGSCs): cells derived from germ cells in the mouse testis that behave similarly to ESCs in culture. Pluripotency: the potential to differentiate into cells of any of the three germ layers (endoderm, mesoderm, or ectoderm). Pluripotency transcription factors (TFs): TFs such as OCT4 (also known as POU5F1), SOX2, and NANOG, which were identified as principal regulators of pluripotency. They are supported by the Kru ¨ ppel transcription factor KLF-4 and also by c-MYC. RNA-binding protein (RBP): a protein that can bind to RNA, usually via a specific RNA-binding domain(s). Self-renewal: the ability to maintain an undifferentiated state while undergoing multiple cycles of cell division. Teratocarcinoma: a mixed germ-cell tumor composed of teratoma and embryonal carcinoma. Teratocarcinomas occur mostly in the testis. Teratoma: a tumor made of different types of tissues that are not native to the location in which it occurs. Teratomas are usually found in the ovary. Corresponding author: Ciosk, R. (rafal.ciosk@fmi.ch). Keywords: pluripotency; DGCR8; LIN28; let-7; GLD-1; DND1. 0168-9525/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tig.2012.10.007 Trends in Genetics, February 2013, Vol. 29, No. 2 99