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    • Using both standard RNA seq and

    2018-11-08

    Using both standard RNA-seq and RNA-seq Sulfo-NHS-Biotin analysis, we identified UPF1-regulated transcripts that are good candidates to be direct NMD targets in hESCs. There was considerable overlap between these candidate NMD target transcripts expressed in hESCs with previously identified high-confidence NMD substrates (Figures 2C and S2A; Table S5). We regarded this overlap as remarkable, given that few high-confidence NMD substrates have thus far been defined in the field and most have been identified in cell lines that bear little resemblance to hESCs (e.g., HeLa and HEK). The overlap was particularly surprising given that NMD has been shown to exhibit tissue specificity (Huang et al., 2011; Karam et al., 2013). We suggest that the high-confidence NMD substrate database we compiled (Figure 2B and Table S5) is a valuable resource for the field. The mRNAs identified as significantly upregulated or significantly stabilized upon UPF1 depletion in hESCs were enriched in several GO categories related to differentiation and morphogenesis (Figure 2D). We note, however, that many of these putative NMD substrates require further assays to determine whether they are bona fide direct NMD targets. The identification of direct NMD targets is clouded by the fact that known “NMD-inducing features” do not necessarily trigger NMD. For example, while long 3′ UTRs are a well-established NMD-inducing feature, many long 3′ UTRs do not trigger NMD (Lykke-Andersen and Jensen, 2015). Indeed, elements have been identified in long 3′ UTRs that permit NMD evasion (Toma et al., 2015), and short 3′ UTRs (<1 kb) can also trigger NMD (Eberle et al., 2008; Hogg and Goff, 2010; Singh et al., 2008; Yepiskoposyan et al., 2011), consistent with the recent finding that candidate NMD substrates in mESCs have 3′ UTRs with shorter average length than control RNAs (Hurt et al., 2013). Further empirical studies will be required before algorithms can be created to identify high-confidence NMD substrates in silico. The ability of NMD to influence cell fate is not restricted to ESCs. For example, we previously showed that high NMD magnitude promotes epidermal cell fate and represses neural cell fate (Lou et al., 2014). NMD represses neural fate by promoting the decay of the mRNA encoding the pro-neural differentiation factor SMAD7 (Lou et al., 2014). How NMD stimulates epidermal cell fate is not known. One possibility is that NMD achieves this through its ability to stimulate BMP signaling (Figures 5 and S4C), as epidermal differentiation requires BMP signaling (Bier and De Robertis, 2015). Our evidence that NMD degrades transcripts encoding a wide array of signaling factors (Figures 2E, 3A, S2B, S2D, and S3) raises the possibility that NMD serves as a regulator of many other cell-fate decisions during development. One subject of future investigation is to examine the role of NMD in the differentiation of primary germ layers at stages following those we examined in this report. Given the diverse roles of NMD in differentiation, it is not surprising that recent evidence suggests that NMD is also involved in malignancy (Chang et al., 2016; Liu et al., 2014; Wang et al., 2011), including through altered TGF-β signaling (Chang et al., 2016). Future studies will be required to elucidate the full complement of developmental events and diseases influenced by NMD.
    Experimental Procedures
    Author Contributions
    Acknowledgments This work was supported by the NIH (GM111838 and HD001259) and CIRM (RB4-06345). The authors thank Thomas Touboul (UCSD) and Maike Sander (UCSD) for their advice with hESC differentiation techniques, and Jens Lykke-Andersen (UCSD) for antibodies.
    Introduction Human pluripotent stem cells (hPSCs) provide a unique resource for basic as well as translational research. Both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are widely used to study early human development (Zhu and Huangfu, 2013), assess the toxic effects of chemicals (Dreser et al., 2015; Zimmer et al., 2012), model human diseases or cancer (Bellin et al., 2012; Funato et al., 2014; Merkle and Eggan, 2013), and discover novel potential drugs (Lee et al., 2012). Furthermore, access to greatly improved protocols for lineage-specific differentiation has led to the first experimental applications of hPSC-derived lineages in regenerative medicine such as in patients with macular degeneration (Schwartz et al., 2015). Other hPSC-based applications that are being pursued intensely include the replacement of hormone-producing cells such as in type 1 diabetes (Pagliuca et al., 2014; Rezania et al., 2014). Replacing hormone-producing cells is a particularly attractive approach for cell therapy, especially if restoration of feedback mechanisms with subsequent dynamic release of hormones can be achieved by the grafted cells.