Control of gene expression in space and time plays an important role in enabling cells to know where they are in the developing embryo and what to become, a process often referred to as cellular specification. Decades of research have demonstrated numerous layers of regulation in control of gene expression, at both the transcriptional and post-transcriptional level, which coordinate this process. Translational contro of gene expression has, on the contrary, received less experimental attention. Most notably, the prevailing dogma is that at the level of protein production, the ribosome -although an immensely complex molecular machine- possesses a constitutive rather than regulatory function in translating mRNAs. Our findings unexpectedly reveal that fundamental aspects of embryonic development and tissue patterning are instead controlled by a highly regulatory function of the ribosome. Importantly, we have shown that specialized ribosomes harboring a unique composition or activity are critically required for the formation of the mammalian body plan and direct where and when key developmental regulators, such as Hox genes, are expressed. In addition, our research has identified novel RNA regulons embedded within the 5'UTRs of key developmental regulators that confer greater gene regulatory potential by the ribosome. In this proposal we will undertake a highly multidisciplinary approach to systematically and comprehensively define for the first time how ribosome composition changes during cell differentiation. Specifically, we hypothesize that the composition of the ribosome is highly dynamic during stem cell differentiation to confer novel regulatory potential to post-transcriptional gene expression underlying rapid and dynamic cell fate decisions. In this proposal we will address this hypothesis through two specific aims.
In Aim 1 we will employ state-of-the-art mass spectrometry to systematically examine ribosome composition in embryonic stem cells and during their differentiation into neural progenitors and cardiomyocytes to functional characterize the contributions of ribosome heterogeneity towards gene regulation.
In Aim 2 we will undertake a highly functional approach to define the repertoire of transcripts that selectively rely on specific ribosome components for their accurate expression during cell fate specification. Together, these studies will bring to bear a new level of regulatory specificit for control of gene expression that instructs cell fate decisions reflecting a paradigm-shift in th regulatory circuitry underlying cell fate specification.

Public Health Relevance

A major challenge in stem cell biology is to understand how gene expression is regulated to give rise to the remarkable diversity of cell type's characteristic of embryonic development. Our findings unexpectedly uncover that fundamental aspects of gene expression and formation of the mammalian body plan are controlled by different types of ribosomes, the protein synthesis machinery of the cell. Here we will characterize how composition of the ribosome machinery changes during stem cell differentiation and how this level of regulation guides stem cell self-renewal and cell fate specification. This knowledge will be vital for understanding the molecular basis of a vast number of human diseases and birth defects associated with mutations in specific ribosome components.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Exploratory/Developmental Grants (R21)
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Development - 2 Study Section (DEV2)
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Mukhopadhyay, Mahua
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Stanford University
Anatomy/Cell Biology
Schools of Medicine
United States
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Leppek, Kathrin; Das, Rhiju; Barna, Maria (2018) Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them. Nat Rev Mol Cell Biol 19:158-174
Simsek, Deniz; Barna, Maria (2017) An emerging role for the ribosome as a nexus for post-translational modifications. Curr Opin Cell Biol 45:92-101
Shi, Zhen; Fujii, Kotaro; Kovary, Kyle M et al. (2017) Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide. Mol Cell 67:71-83.e7
Simsek, Deniz; Tiu, Gerald C; Flynn, Ryan A et al. (2017) The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity. Cell 169:1051-1065.e18