Regulation of ES cell differentiation by NMD

Mammalian development proceeds through a series of cell fate transitions, typically accompanied by a decline in developmental potency. Research in recent years has contributed to a substantial understanding of the molecular underpinnings of pluripotency. However, the mechanisms that compute cues from the cellular environment to elicit a regulated and exact cell fate choice are currently unknown. As a consequence, primary lineage decisions from mammalian pluripotent cells cannot be properly controlled experimentally. Work in my lab focuses on identifying and studying molecular processes that drive and regulate proper differentiation. We have identified a cohort of such processes that regulate ES cell differentiation in a genome-wide screen in haploid ES cells. One such process is nonsense mediated mRNA decay (NMD). NMD maintains cellular homeostasis by eliminating target RNAs that contain premature termination codons. Thereby it constitutes a cellular surveillance mechanism that degrades transcripts encoding for potentially deleterious proteins. To study NMD function we have generated a series of ES cell lines deficient for a several NMD factors. They all show a clear defect in differentiation without affecting self-renewal and ESC identity. Although significant redundancy and parallel activity has been proposed for different NMD degradation pathways, we find clear and reproducible differences both in phenotypic strength as well as target gene deregulation between different NMD factor knockout cell lines. The downstream target genes of NMD that are actually causative for the observed differentiation defects in NMD deficient ES cells are unknown. To identify those we will follow a multilayered approach. We will use state-of-the-art technological approaches including analysis of RNA metabolism, CRISPR based genome-engineering and genome-wide suppressor screens to identify central regulatory targets of NMD that contribute to the observed differentiation defects. Taken together, our results will advance the understanding of the molecular underpinnings of early embryonic cell fate decisions. We will translate detailed molecular findings made in mouse cell culture both to in vivo development as well as to human ESC differentiation. In addition, the ability to culture homogeneous mES cell cultures, combined with genetic and biochemical accessibility will provide a potent platform to advance our understanding of how nonsense mediated mRNA decay is regulated and how NMD components differentially regulate target mRNA degradation and transcription states.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Our work shows that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.