The RNA–Methyltransferase Misu (NSun2) Poises Epidermal Stem Cells to Differentiate Sandra Blanco1, Agata Kurowski1, Jennifer Nichols1, Fiona M. Watt1,2, Salvador Aznar Benitah 3 , Michaela Frye1* 1 Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, United Kingdom, 2 CR-UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom, 3 Center for Genomic Research (CRG) and UPF, Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA), Barcelona, Spain Abstract Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post- transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/ Nsun2) is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in enhanced quiescence and aberrant stem cell differentiation. Our results reveal that post-transcriptional RNA methylation can play a previously unappreciated role in controlling stem cell fate. Citation: Blanco S, Kurowski A, Nichols J, Watt FM, Benitah SA, et al. (2011) The RNA–Methyltransferase Misu (NSun2) Poises Epidermal Stem Cells to Differentiate. PLoS Genet 7(12): e1002403. doi:10.1371/journal.pgen.1002403 Editor: Tony Oro, Stanford University, United States of America Received March 29, 2011; Accepted October 17, 2011; Published December 1, 2011 Copyright: 2011 Blanco 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: This work was funded by Cancer Research UK (C10701/A7006) and the Medical Research Council (MRC) (89372). SB was supported by Ramo´ n Areces Spanish Foundation and EMBO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Misu is part of a current patent application. * E-mail: Michaela.Frye@cancer.org.uk Introduction Stem cells are defined by their ability to continuously maintain their population (self-renewal) while generating progeny (differen- tiation). During self-renewal, stem cells have to avoid cell cycle exit and differentiation; however, when differentiating they have to evade uncontrolled proliferation. Thus, the question of how the balance between self-renewal and commitment is regulated is highly relevant to a fundamental understanding of cell differen- tiation and cancer. The hair follicle offers an excellent model to study stem cell fate, as it undergoes cyclic bouts of growth (anagen), apoptosis- mediated regression (catagen) and rest (telogen) [1]. Multipotent hair follicle stem cells, located in a special microenvironment called the bulge, are slow cycling but exhibit long-term contribution to all hair compartments [2,3]. At the early onset of hair growth, single bulge cells migrate out of their niche in telogen and undergo proliferation as progenitors before they differentiate into hair [4,5]. Once a stem cell has left its niche, intrinsic and environmental cues converge to balance proliferation of progenitors with lineage- specific differentiation. For example, c-Myc is known to control the balance between stem cell expansion and differentiation [6,7,8]. When activated in epidermal stem cells, Myc triggers their exit from the stem cell compartment, induces proliferation of progenitors, and subsequently leads to lineage-specific differenti- ation [9,10,11]. Because the nucleolar protein Misu/NSun2 (Myc-induced SUN- domain-containing protein) is direct target gene of c-Myc [12], we considered the possibility that its RNA methyltransferase activity may represent a novel mechanism to regulate stem cell fate. Misu catalyzes the formation of 5-methylcytidine (m5C) in tRNA and possibly other RNA species [12,13,14]. Whereas the function of (cytosine-5) methylated DNA has been extensively analyzed [15], the functional roles of methylated RNA are largely unknown [16]. To date, Misu (NSun2) is one of only two identified m5C methylases with substrate specificity towards tRNA [13,17,18,19]. Here, we demonstrate that Misu is required for normal tissue homeostasis in vivo. Expression of Misu is up-regulated in the hair follicle bulge at the entry of anagen. Deletion of Misu prolongs stem cell quiescence leading to a delay in initiation of anagen. Thus, our data reveals a post-transcriptional modification as a novel mechanism used by stem cells to precise and temporally accurate balance self-renewal and differentiation. Results Ablation of Misu blocks tRNA methylation and causes weight loss and partial alopecia To functionally analyse the role of Misu in vivo, we generated a reporter-tagged loss-of-function mouse model using an ES cell line carrying a Gene Trap in intron 8 of the NSUN2 gene (GGTC- clone ID: D014D11). The methyltransferase activity of Misu is mediated by the conserved SUN domain encoded by exon 2 to 12 (Figure S1A; red box) [19]. Insertion of the Gene Trap and fusion to the reporter b-galactosidase leads to disruption of the catalytic domain of Misu (Figure S1A; blue box) [20]. We confirmed deletion of Misu and presence of b-galactosidase by gene-specific PCR (Figure S1B). Quantitative real-time PCR (QPCR) using a probe for NSun2 showed substantial reduction of full-length Misu in skin of heterozygous (+/2) and loss of expression in homozygous mice (2/2) (Figure S1C). Western Blot analysis demonstrated that Misu 2/2 mice lacked Misu protein (Figure S1D). PLoS Genetics | www.plosgenetics.org 1 December 2011 | Volume 7 | Issue 12 | e1002403