Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway.

We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.

The Rac and Cdc42 GTPases share several regulators and effectors, yet perform distinct biological functions. The factors determining such specificity in vivo have not been identified. In a mutational screen in Drosophila to identify Rac-specific signaling components, we isolated 11 alleles of myoblast city (mbc). mbc mutant embryos exhibit defects in dorsal closure, myogenesis, and neural development. DOCK180, the mammalian homolog of Mbc, associates with Rac, but not Cdc42, in a nucleotideindependent manner. These results suggest that Mbc is a specific upstream regulator of Rac activity that mediates several morphogenetic processes in Drosophila embryogenesis.Received April 22, 1998; revised version accepted September 2, 1998.The Rho family of GTPases, which includes Rho, Rac, and Cdc42, regulate a variety of cellular processes including cytoskeletal reorganization, endocytosis, cell cycle progression, and transcriptional activity (for review, see van Aelst and D'Souza-Schorey 1997). In fibroblasts, activation of Cdc42, Rac, or Rho leads to particular rearrangements of the actin cytoskeleton resulting in filopodia, lamellipodia, and stress fiber formation, respectively. Moreover, evidence suggests that each of these GTPases can be activated by distinct extracellular stimuli. Several proteins have now been identified that interact selectively with either Rho, Rac, or Cdc42, and it seems likely that the specificity of these interactions in vitro accounts for at least some of the signaling specificity among these GTPases in vivo (Hall 1998). However, the various Rho family members have also been found to share several regulators and effector targets in in vitro studies (Hall 1998), thereby complicating a thorough mechanistic understanding of the signaling specificity that is observed in vivo.In Drosophila, distinct requirements for closely related homologs of the Rho, Rac, and Cdc42 GTPases have been identified in the various morphogenetic events associated with embryogenesis. For example, Rho1 is required for gastrulation (Barrett et al. 1997); Rho1, Rac1, and Cdc42 are required for dorsal closure and tissue polarity (Harden et al. 1995;Eaton et al. 1996;Strutt et al. 1997); and Rac1 and Cdc42 have been implicated in neural development and myogenesis (Luo et al. 1994). Using Drosophila genetics, we identified Myoblast city (Mbc), a homolog of mammalian DOCK180 (Erickson et al. 1997) and Caenorhabditis elegans CED-5 (Wu and Horvitz 1998) as a specific mediator of Rac1 activity in several morphogenetic processes during Drosophila embryogenesis, including myogenesis, neural development, and dorsal closure. Results and Discussion Overexpression of Rho, Rac, and Cdc42 GTPases in the fly eye causes distinct developmental defectsPreviously, we described developmental eye defects caused by overexpression of the wild-type Drosophila Rho1 GTPase in transgenic flies (Hariharan et al. 1995).To determine whether the related Rac and Cdc42 GTPases could similarly disrupt eye development, we generated transgenic flies in which w...

Characterization of signaling pathways in embryonic stem cells is a prerequisite for future application of these cells to treat human disease and other disorders. Identification of tyrosine signaling cascades is of particular interest but is complicated by the relatively low levels of tyrosine phosphorylation in embryonic stem cells. These hurdles correlate with the primary limitations of mass spectrometry-based proteomics; namely, poor detection limit and dynamic range. To overcome these obstacles, we fabricated miniaturized LC-electrospray assemblies that provided approximately 15-fold improvement in LC-MS performance. Significantly, our characterization data demonstrate that electrospray ionization efficiency compensates for diminished chromatographic performance at effluent flow rates below Van Deemter minima. Use of these assemblies facilitated quantitative proteomics-based analysis of tyrosine signaling cascades in embryonic stem cells. Our results suggest that a renewed focus on miniaturized LC coupled to ultralow flow electrospray will provide a viable path for proteomic analysis of primary cells and rare post-translational modifications.

No abstract

The PKN family of PKC-related protein kinases constitutes the major Rho GTPase-associated protein kinase activities detected in mammalian tissues. However, the biological functions of these kinases are unknown. We have identified a closely related PKN homolog in Drosophila (Pkn) that binds specifically to GTP-activated Rho1 and Rac1 GTPases through distinct binding sites on Pkn. The interaction of Pkn with either of these GTPases results in increased kinase activity, suggesting that Pkn is a shared Rho/Rac effector target. Characterization of a loss-of-function mutant of Drosophila Pkn revealed that this kinase is required specifically for the epidermal cell shape changes during the morphogenetic process of dorsal closure of the developing embryo. Moreover, Pkn, as well as the Rho1 GTPase, mediate a pathway for cell shape changes in dorsal closure that is independent of the previously reported Rac GTPase-mediated Jun amino (N)-terminal kinase (JNK) cascade that regulates gene expression required for dorsal closure. Thus, it appears that distinct but coordinated Rho-and Rac-mediated signaling pathways regulate the cell shape changes required for dorsal closure and that Pkn provides a GTPase effector function for cell shape changes in vivo, which acts together with a Rac-JNK transcriptional pathway in the morphogenesis of the Drosophila embryo.

Advances in chemistry and massively parallel detection underlie DNA sequencing platforms that are poised for application in personalized medicine. In stark contrast, systematic generation of protein-level data lags well-behind genomics in virtually every aspect: depth of coverage, throughput, ease of sample preparation, and experimental time. Here, to bridge this gap, we develop an approach based on simple detergent lysis and single-enzyme digest, extreme, orthogonal separation of peptides, and true nanoflow LC-MS/MS that provides high peak capacity and ionization efficiency. This automated, deep efficient peptide sequencing and quantification (DEEP SEQ) mass spectrometry platform provides genome-scale proteome coverage equivalent to RNA-seq ribosomal profiling and accurate quantification for multiplexed isotope labels. In a model of the embryonic to epiblast transition in murine stem cells, we unambiguously quantify 11,352 gene products that span 70% of Swiss-Prot and capture protein regulation across the full detectable range of high-throughput gene expression and protein translation.

Summary Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG-binding protein MBD2, whose isoforms play opposing roles in maintenance of, and reprogramming to, pluripotency. While both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity complexity in support of hPSC self-renewal and reprogramming.