A Latent Pro-Survival Function for the Mir-290-295 Cluster in Mouse Embryonic Stem Cells Grace X. Y. Zheng1,2,3.¤a, Arvind Ravi1,3,4., J. Mauro Calabrese1,3¤b, Lea A. Medeiros3,5, Oktay Kirak5, Lucas M. Dennis3,5¤c, Rudolf Jaenisch3,5, Christopher B. Burge2,3, Phillip A. Sharp1,3* 1 MIT Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, United States of America, 2 Computational and Systems Biology Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 4 Harvard-MIT Health Sciences and Technology Program, Cambridge, Massachusetts, United States of America, 5 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America Abstract MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir- 290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target. Citation: Zheng GXY, Ravi A, Calabrese JM, Medeiros LA, Kirak O, et al. (2011) A Latent Pro-Survival Function for the Mir-290-295 Cluster in Mouse Embryonic Stem Cells. PLoS Genet 7(5): e1002054. doi:10.1371/journal.pgen.1002054 Editor: Michael T. McManus, University of California San Francisco, United States of America Received January 29, 2011; Accepted March 6, 2011; Published May 5, 2011 Copyright: 2011 Zheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: AR was funded by a Fannie and John Hertz Foundation Fellowship. This work was supported by NIH grant RO1-GM34277, NCI grant PO1-CA42063, and the NCI Cancer Center Support (core) grant P30-CA14051. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sharppa@mit.edu . These authors contributed equally to this work. ¤a Current address: Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford School of Medicine, Stanford, California, United States of America ¤b Current address: Department of Genetics and the Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina, United States of America ¤c Current address: NanoString Technologies, Seattle, Washington, United States of America Introduction MicroRNAs (miRNAs) are endogenous ,22 nt RNAs that regulate gene expression post-transcriptionally. In animals, the ability of miRNAs to accomplish this regulation depends on complementarity between mature miRNA sequences and their mRNA targets. Most commonly, partial binding of miRNAs leads to destabilization of mRNA transcripts and/or inhibition of productive translation, and in rare cases perfect complementarity instead causes target cleavage. Both in vitro experiments and bioinformatics have shown that matches to positions 2–7 of the miRNA, referred to as the miRNA ‘‘seed,’’ are generally required for effective miRNA-directed mRNA downregulation [1,2]. The roles of miRNAs in mouse embryonic stem cells (mESCs) have been of particular interest, as this knowledge may shed light on key aspects of mammalian development and generate useful insights into reprogramming and cancer, both of which recapit- ulate aspects of an ESC expression state [3,4]. In addition, the survival of mESCs in the absence of Dicer (Dcr), the key RNase III enzyme that generates mature miRNAs, makes them a unique model system for dissecting miRNA function [5,6]. Several large- scale sequencing datasets [7,8,9] have revealed that the mir-290- 295 cluster constitutes the dominant miRNA population in mESCs, giving rise to about 50% of all reads in these cells (Table S1). Many of the miRNAs in this cluster share the hexamer seed ‘AAGUGC,’ which is also expressed at much lower levels by the mir-302 and mir-467 clusters, contributing less than 5% of total reads (Table S2). A similar percent contribution to total miRNA levels comes from the miR-17-92 family, which contains the shifted seed ‘AAAGUG,’ and therefore may share some common targets (Table S2) [7,8,9]. Given the abundance of the mir-290- 295 cluster and these related sequences, much of mESC miRNA physiology is likely to be a function of this dominant seed sequence. Within the mir-290-295 cluster, the ‘AAGUGC’ seed is found in miR-290-3p, miR-291a-3p, miR-291b-3p, miR-292-3p, miR-294, and miR-295. Consistent with their high expression, these miRNAs (which we shall refer to as the mir-295 cluster) have PLoS Genetics | www.plosgenetics.org 1 May 2011 | Volume 7 | Issue 5 | e1002054