Specialized Translational Control of Stem Cell Differentiation and Embryonic Development

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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 control of gene expression has, on the contrary, received less experimental attention. Most notably, the prevailing dogma has been 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 have established a new field of study by demonstrating that ribosomes are highly regulatory in control of the expression of developmental gene regulatory networks underlying tissue patterning and formation of the mammalian body plan. In our most recent studies, we have identified entire biological pathways in embryonic stem cells represented by the translational preferences of specific ribosomes, that differ in the composition of their ribosomal proteins (RPs) or the interaction of novel ribosome-associated proteins (RAPs) that we have recently identified that directly associate with mammalian ribosomes. We have further shown ribosome heterogeneity in proximity to key cellular organelles as a mechanism to control localized protein production within subcellular space. These findings change our understanding of gene regulation and open a new portal of study into an additional layer of gene expression vital to control of cell specification, tissue patterning, and embryonic development. In this proposal we will undertake a highly multidisciplinary approach to characterize this novel regulatory code for translational control of the circuitry of key developmental networks. In Aim1 we will extend our new roadmap of ribosome heterogeneity indicated by the presence of distinct ribosomes during primary human ES cellular differentiation to an organismal level. In particular, we will leverage novel genetic tools to study ribosome biology in-vivo. Using this approach, we will delineate the mechanisms by which a single RP can control a paramount step in embryonic development, namely sustained paraxial mesoderm formation, and its role in translational control of the WNT signaling pathway, which reflects a novel step in the regulation of a major signaling pathway in development. In Aim2 we will undertake a systems level approach to characterize the role of ribosomes as key regulators of cell fate transitions. We will utilize novel technologies to forcibly and inducibly remove specific RPs selectively from cytoplasmic ribosomes for the first time and assess their individual functions on stem cells differentiation down the mesoderm and endoderm lineages. In Aim3 we will functionally characterize alternative RP paralogs in mammary-glad development for which our compelling preliminary data indicate that they translate distinct subsets of mRNAs during the switch to lactation. We hypothesize that translation control is required to synthesize copious milk proteins critical for neonate sustenance. Defining at a more basic level the specificity and dynamics of ribosome-mediated control gene regulation will be invaluable for our understanding of how deregulations in the ribosome alter accurate control of gene expression underlying human congenital birth defects.