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    • br Experimental Procedures br Author Contributions br Acknow

    2018-10-24


    Experimental Procedures
    Author Contributions
    Acknowledgments This work was supported by JST, CREST and MEXT (to E.N.), and MEXT Scholarship Program (to J.L.).
    Introduction Developmental processes depend on highly orchestrated shifts in the levels of specific mRNAs. While regulation of the synthesis of mRNAs (transcription) has traditionally been the focus of attention, increasing evidence suggests that developmentally regulated alterations in the rate of decay of specific transcripts also influences developmental decisions (Hwang and Maquat, 2011). The best-studied RNA degradation pathway is nonsense-mediated RNA decay (NMD). While originally identified as a quality control mechanism that rapidly degrades aberrant transcripts derived from mutant genes, NMD was subsequently found to also degrade many normal transcripts (Peccarelli and Kebaara, 2014). Between ∼3% and 20% of the transcriptomes of eukaryotic organisms ranging from yeast to man are regulated (directly or indirectly) by NMD (Peccarelli and Kebaara, 2014). The specific transcripts targeted by NMD are those that harbor a stop prolyl hydroxylase inhibitor in a “premature” context, as this leads to the formation of a complex of NMD proteins that subsequently recruits RNA decay factors (Schoenberg and Maquat, 2012). The discovery that NMD regulates normal gene expression raised the possibility that NMD can influence normal biological events (Hwang and Maquat, 2011). By modulating the magnitude of NMD, batteries of transcripts can be stabilized or destabilized to achieve specific biological outcomes. Indeed, NMD has been found to be a highly regulated pathway, and mounting evidence supports the possibility that NMD is critical for many biological events, with loss of NMD resulting in developmental defects (Huang and Wilkinson, 2012; Hwang and Maquat, 2011; Karam et al., 2013). Most well-studied is the role of NMD in the neural cell lineage. Studies in Drosophila melanogaster, zebrafish, and mammalian cell lines have shown that NMD is critical for specific steps in neural development (Jolly et al., 2013; Lou et al., 2014; Metzstein and Krasnow, 2006; Wittkopp et al., 2009). In humans, mutations in the NMD gene, UPF3B, lead to intellectual disability (Nguyen et al., 2014). These cognitive disorders are likely to result from developmental defects; indeed patients with mutations in UPF3B and copy-number variants of other NMD genes commonly have neurodevelopmental disorders, including schizophrenia and autism (Nguyen et al., 2014). Less is known about the influence of NMD on non-neuronal cell lineages. Loss of the NMD factor UPF2 disrupts hematopoiesis and liver development in vivo (Thoren et al., 2010; Weischenfeldt et al., 2008), and evidence suggests that NMD cooperates with another RNA decay pathway to influence muscle cell differentiation (Gong et al., 2009). While these studies strongly suggest that NMD has roles in various developmental systems, the underlying mechanism is poorly understood. Here we examine the role of NMD in the differentiation of human embryonic stem cells (hESCs). This was motivated by earlier work suggesting that NMD functions in early embryogenesis: null mutations in four NMD genes—Upf1, Upf2, Smg1, and Smg6—result in early embryonic lethality in mice (Li et al., 2015; McIlwain et al., 2010; Medghalchi et al., 2001; Weischenfeldt et al., 2008). These studies raised the possibility that NMD is critical for very early embryonic developmental events in mammals, a possibility we investigate in hESCs. Our studies reveal a role for NMD in definitive endoderm and mesoderm lineage segregation through the ability of NMD to regulate the levels of mRNAs encoding signaling pathway factors.
    Results
    Discussion In this study, we provide several lines of evidence that the highly conserved RNA degradation pathway, NMD, strongly influences hESC differentiation. Through marker analysis, we found that NMD promotes the differentiation of hESCs into mesoderm and inhibits their differentiation into definitive endoderm (Figures 1E–1H, 3, 4, and 5). Our results suggest that NMD magnitude is critical for dictating the proportion of cells that progress down these two cell lineages by regulating the balance between TGF-β and BMP signaling (Figure 6). We obtained several lines of evidence that NMD inhibits TGF-β signaling and activates BMP signaling (Figures 3, 4, 5, S3, and S4), which are known to promote endoderm and mesoderm differentiation, respectively (Loh et al., 2014). Thus, by altering NMD magnitude, the relative strength of these two signaling pathways is shifted, thereby influencing cell fate (Figure 6). Recent studies have identified an array of modulatory factors that shift NMD magnitude, including microRNAs, eIF2α phosphorylation, RNA-binding proteins, and the level of specific NMD factors (Ge et al., 2016; Huang and Wilkinson, 2012; Karam et al., 2013; Shum et al., 2016). We suggest that one or more of these mechanisms may be responsible for altering the magnitude of NMD in hESCs to influence cell fate.