ace inhibitor br Results br Discussion The

Results
Discussion The length of poly(A) tails plays an important role in controlling mRNA degradation and translational silencing, and is therefore dynamically regulated during development and disease (Eckmann et al., 2011; Norbury, 2013). A recent study showed that poly(A) tail length predominantly correlated with mRNA stability in transcriptionally active cells. Furthermore it showed that, during embryonic development, poly(A) tail length initially regulated translational efficiency and gradually shifted to regulate mRNA stability as zygotic transcription started (Subtelny et al., 2014). Our results are consistent with the above observations, and suggest that poly(A) tail-length regulation by Ccr4-Not may start to affect mRNA stability as early as at the ace inhibitor stage in mammals. The Ccr4-Not complex is one of the main deadenylases in mammalian cells that remove poly(A) tails. It can catalyze deadenylation in both gene- and context-specific manners to allow control of poly(A) tail lengths (Collart and Panasenko, 2012; Shirai et al., 2014; Xu et al., 2014). It has been proposed that different RNA binding proteins and/or different subunits in the Ccr4-Not complex may mediate the recruitment and regulation of different target mRNAs. For example, in germ cell development, NANOS2 and NANOS3 can interact with different Ccr4-Not subunits to regulate different mRNA targets (Suzuki et al., 2014). In ESCs, PUM1 facilitates the exit from the pluripotent state by binding to pluripotency gene mRNAs and promoting their degradation (Leeb et al., 2014), presumably via the interaction with CNOT7/8 in the Ccr4-Not complex (Van Etten et al., 2012). Thus, we propose that there may exist specific RNA binding proteins that facilitate the regulation of differentiation gene mRNAs by CNOT3 and Ccr4-Not in pluripotent cells. This is consistent with the “RNA Regulon” hypothesis, which suggests that mRNAs encoded by functionally related genes may be coordinately regulated by specific RNA processing machineries (Keene, 2007). Along similar lines, it was recently reported that m6A mRNA methylation reduces pluripotency gene mRNA stability to facilitate the exit from the pluripotent state in ESCs and that m6A depletion led to post-implantation lethality (Geula et al., 2015). Although it is not clear whether Ccr4-Not is involved in the m6A-mediated mRNA regulation, these and our results highlight the critical role of post-transcriptional regulation in the maintenance of the pluripotent state both in vitro and in vivo. Finally, we found that CNOT3 target gene mRNAs have shorter half-lives in ESCs and acquire longer half-lives during differentiation. This result suggested that mRNA stability may indeed be a critical regulatory step, and that CNOT3, as well as Ccr4-Not, plays an important role in such a regulation to control pluripotent cell-fate specification. In addition, we found that CNOT3 target genes possess both active and repressive histone markers at their promoters. Promoter regions with bivalent histone modifications were initially discovered in ESCs at developmental genes, and were later shown to be present in epiblast cells during mouse development (Voigt et al., 2013). These bivalent genes are transcriptionally poised and can be activated upon suitable developmental cues to facilitate the exit from pluripotency. We propose that in addition to the transcriptional repression, differentiation genes may be further silenced by the Ccr4-Not complex at the mRNA level. Regulation of mRNA poly(A) tail length and stability may provide a quick and potentially reversible means to influence differentiation gene expression. Thus, by controlling the timely expression of differentiation genes, CNOT3 plays an essential role in maintaining the responsiveness of pluripotent cells to developmental signals.
Experimental Procedures
Author Contributions
Acknowledgments We thank Dr. Anna-Katerina Hadjantonakis for helpful discussions. We thank the NIEHS Animal, Epigenomics, Bioinformatics, Protein Expression, Imaging, and Histology core facility for assistance with various techniques and experiments. This study was supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health SciencesZ01ES102745 (to G.H.), Z01ES102985 (to C.J.W.), and Z01ES102625 (to R.J.).