Author Summary We demonstrate that the RNA methyltransferase activity of Misu/NSun2 is required for the proper maintenance of the epidermal differentiation program, and thus post-tran- scriptional mechanisms are involved in controlling the balance between stem cell self-renewal and differentiation. Heterozygous mice for Misu deletion were viable and did not display an obvious phenotype (Figure 1A). Homozygous mice were also viable, but were appreciably smaller than their wild type (wt) and heterozygous littermates (Figure 1A). At three months of age, Misu 2/2 mice weighed around 30% less than controls (Figure 1B). The smaller size of Misu 2/2 mice was confirmed by a second knock-out mouse model Nsun2tm1a(EUCOMM)Wtsi generated by the Wellcome Trust Sanger Institute (http://www. sanger.ac.uk) (data not shown). Misu 2/2 males were sterile, and both genders had pelage that was comparable to controls after Figure 1. Loss of Misu results in block of tRNA methylation and causes reduced body weight and partial alopecia. (A) Gross phenotype of wild type, Misu +/2 and 2/2 neonates. (B) Average weight of Misu 2/2 mice is reduced compared to controls. (C,D) Pelage of 10 months old wild-type (C) and Misu 2/2 mouse (D). Error bars represent standard deviation (SD). Wt: wild-type; +/2: heterozygous mice; 2/2: homozygous mice. (E) Bisulfite sequencing of intron- containing tRNALeu (CAA) from wild-type and Misu 2/2 mouse skin. The diagram shows the methylation status of tRNALeu (CAA) transcribed from two different genes: tRNA-988Leu (CAA) (top panel) and gene tRNA- 1911Leu (CAA) (bottom panel). Black boxes indicate methylated cytosine residues and white boxes indicate unmethylated cytosine residues. Numbers above boxes indicate the cytosine positions in the primary tRNA sequence amplified (nucleotides 29–36+intron sequence). Asterisk indicates the position of the expected methylated cytosine. Numbers+I indicate the cytosine positions in the intron sequence. doi:10.1371/journal.pgen.1002403.g001 Misu Regulates Stem Cell Fate birth, but showed cyclic alopecia at around 10 months of age (Figure 1C, 1D). Both, the yeast and human orthologue of Misu have previously been shown to catalyse the methylation of cytosine at position 34 (C34) in intron-containing pre-tRNALeu (CAA) [13,19]. To inves- tigate whether mouse Misu also methylated tRNALeu (CAA), we used RNA bisulfite sequencing, which allows analysis of RNA methylation patterns in their native sequence context [21]. We find that intron-containing pre-tRNALeu (CAA) was methylated at C34 in wild-type mice but lacked the modification when Misu was deleted (Figure 1E). We concluded that Misu was indispensable for methylation of tRNALeu (CAA) in skin. Based on our observation that older mice show signs of alopecia in the absence of Misu we speculated that Misu might have a functional role in maintaining skin homeostasis in the long-term. Misu is dynamically expressed in embryogenesis and adult skin To determine the role of Misu in skin, we began by examining endogenous expression of Misu during embryogenesis and skin development by staining for LacZ (Figure 2A–2Q). Misu- expression was detected from E3.5 in the inner cell mass of the blastocyst (Figure 2A). After implantation and gastrulation, at E6.5, Misu was observed throughout the extra-embryonic ectoderm (Figure 2B, 2B9), which gives rise to the nervous system and epidermis. Starting from E9.5, expression of Misu became more restricted and at E13.5 and E14.5 Misu was enriched in developing whiskers (arrow) and eyes (arrowhead) (Figure 2C–2F). From E15.5, when the interfollicular epidermis (IFE) begins to stratify and follicular morphogenesis starts by forming hair placodes (Figure 2G), highest expression of Misu was found in the suprabasal layer of IFE (Figure 2G–2I; arrows). After birth, from postnatal day 1 (P1), expression of Misu in the IFE waned (Figure 2J, 2K, 2N, arrows) but increased in the matrix of growing (anagen) hair follicles (Figure 2L, 2M, arrowhead). At the end of follicular morphogenesis, from around P14 to P19, when hair follicles progress through the destructive (catagen) and resting (telogen) phase of the hair cycle, expression of Misu was absent (data not shown and Figure 2N). At early anagen (P23), Misu was up-regulated in the hair germ (HG; arrowhead) and weakly expressed in the bulge (Bu; arrow) (Figure 2O; insert). Throughout anagen, Misu was highly expressed in matrix but was also found in differentiated lineages of the hair follicles (Figure 2P, 2Q, Figure S2). We confirmed the dynamic expression pattern of Misu in skin by detecting endogenous RNA levels of Misu during morphogenesis (M), catagen (C), telogen (T), and anagen (A) of the first postnatal hair cycle by QPCR (Figure 2R). In conclusion, Misu was dynamically expressed during mor- phogenesis and in adult skin. In adult skin, expression of Misu was up-regulated in the bulge and hair germ as soon as the hair follicle entered its growing phase (anagen). During anagen, cells of the hair germ give rise to the hair matrix, which showed highest expression of Misu. Matrix cells, which are often referred as to transit amplifying (TA) cells because they only survive through anagen [22], are stem cell progenitors that divide a finite number of times until they become differentiated. Misu is a transcriptional target of the Lef1/b-Catenin complex in hair follicles Misu was originally identified as a transcriptional target of c- Myc in skin [12]. However, activation of c-Myc triggers epidermal stem cells to differentiate mainly into lineages of IFE and PLoS Genetics | www.plosgenetics.org 2 December 2011 | Volume 7 | Issue 12 | e1002403