Pumilio Proteins Regulate Translation in Embryonic Stem Cells and are Essential for Early Embryonic Development
This is an Open Access Thesis
Abstract
Embryonic stem (ES) cells are defined by their dual abilities to self-renew and to differentiate into any cell type in the body. This vast potential is precisely controlled by spatial and temporal gene regulation at transcriptional, post-transcriptional, and epigenetic levels. Recent studies have revealed several transcription factors that are essential for stem cell self-renewal and pluripotency, but the role of translational control in ES cells is poorly understood. Translational control is a fundamental mechanism of gene regulation during early development, and likely explains the discrepancies between the transcriptome and proteome profiles of stem cells and their differentiated progeny. Pumilio proteins are well-characterized RNA-binding translational repressors that are required for germline stem cell maintenance in Drosophila. However, relatively little is known about the two mammalian Pumilio proteins, Pumilio 1 (Pum1) and Pumilio 2 (Pum2). In this dissertation I characterize the mRNA targets, protein partners, and in vitro and in vivo function of Pum1 and Pum2.Pum1- and Pum2-deficient mouse embryonic stem cell (mES) lines and conditional knockout mice were generated as a means to unravel the function of Pumilio proteins in ES cells and during early development. Pum1-/- and Pum2-/- ES cells grow more slowly than wild type ES cells but remain self-renewing and pluripotent. Pum1-/- and Pum2-/- mice are fertile and viable. Pum1-/- mice are smaller than their littermates, have a hunched appearance that becomes more prominent with age, frequently develop ulcerative dermatitis, and have disorganized, blunted intestinal villi compared to wild type mice. Pum1+/-; Pnm2-/- mice are viable, Pum1-/-; Pum2+/- mice are born alive but have no oral intake and die within 24 hours, and Pum1-/-; Pum2-/- double knockout animals are embryonic lethal by e8.5.Pum1 and Pum2 are highly expressed in the cytoplasm of mES cells. RNA Immunoprecipitation-Microarray (RIP-Chip) analysis of mES lysate reveals that Pum1 binds to 1947 mRNAs and Pum2 binds to 437 mRNAs that comprise almost a complete subset of Pum1 targets. Transcription factors, genes involved in cell cycle control, and genes involved in embryonic patterning are significantly enriched among the mRNA targets of both Pum1 and Pum2. Several targets including Cyclin E, Cyclin B1, and Pum2 are translationally repressed by Pum1, as indicated by changes in protein level without corresponding changes in mRNA level. In mES cells, Pum1 is part of a ∼450 kDa protein complex and Pum2 is part of a ∼350 kDa complex as shown by size exclusion chromatography. Co-immunoprecipitation and mass spectrometry were used to identify three novel binding partners of Pum1: Anaphase-promoting complex subunit 1 (APC1), Regulator of nonsense transcripts 1 (RENT1), and Zinc Finger Protein 198 (ZNF198). Overall, this study reveals an essential function of mammalian Pumilio proteins during early embryogenesis, identifies mRNA targets that are translationally controlled by Pum1 and Pum2 in mES cells, and suggests novel protein-protein interactions that lend insight into the mechanism of action of Pumilio-mediated translational repression.