No abstract

Meiotic progression in Xenopus oocytes, and all other oocytes investigated, is dependent on polyadenylation-induced translation of stockpiled maternal mRNAs. Early during meiotic resumption, phosphorylation of CPE-binding protein (CPEB) is required for polyadenylation-induced translation of mRNAs encoding cell cycle regulators. Xenopus Gef (XGef), a Rho-family guanine-exchange factor, influences the activating phosphorylation of CPEB. An exchange-deficient version of XGef does not, therefore implicating Rho-family GTPase function in early meiosis. We show here that Clostridium difficile Toxin B, a Rho-family GTPase inhibitor, does not impair early CPEB phosphorylation or progression to germinal vesicle breakdown, indicating that XGef does not influence these events through activation of a Toxin-B-sensitive GTPase. Using the inhibitors U0126 for mitogen-activated protein kinase (MAPK), and ZM447439 for Aurora kinase A and Aurora kinase B, we found that MAPK is required for phosphorylation of CPEB, whereas Aurora kinases are not. Furthermore, we do not detect active Aurora kinase A in early meiosis. By contrast, we observe an early, transient activation of MAPK, independent of Mos protein expression. MAPK directly phosphorylates CPEB on four residues (T22, T164, S184, S248), but not on S174, a key residue for activating CPEB function. Notably, XGef immunoprecipitates contain MAPK, and this complex can phosphorylate CPEB. MAPK may prime CPEB for phosphorylation on S174 by an as-yet-unidentified kinase or may activate this kinase.

XGef was isolated in a screen for proteins interacting with CPEB, a regulator of mRNA translation in early Xenopus development. XGef is a Rho-family guanine nucleotide exchange factor and activates Cdc42 in mammalian cells. Endogenous XGef (58 kDa) interacts with recombinant CPEB, and recombinant XGef interacts with endogenous CPEB in Xenopus oocytes. Injection of XGef antibodies into stage VI Xenopus oocytes blocks progesterone-induced oocyte maturation and prevents the polyadenylation and translation of c-mos mRNA; injection of XGef rescues these events. Overexpression of XGef in oocytes accelerates progesterone-induced oocyte maturation and the polyadenylation and translation of c-mos mRNA. Overexpression of a nucleotide exchange deficient version of XGef, which retains the ability to interact with CPEB, no longer accelerates oocyte maturation or Mos synthesis, suggesting that XGef exchange factor activity is required for the influence of overexpressed XGef on oocyte maturation. XGef overexpression continues to accelerate c-mos polyadenylation in the absence of Mos protein, but does not stimulate MAPK phosphorylation, MPF activation, or oocyte maturation, indicating that XGef may function through the Mos pathway to influence oocyte maturation. These results suggest that XGef may be an early acting component of the progesterone-induced oocyte maturation pathway.

Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process.

Ascorbic acid (AsA), an important antioxidant and growth regulator, and it is essential for plant development and human health. Specifically, humans have to acquire AsA from dietary sources due to their inability to synthesize it. The AsA biosynthesis pathway in plants has been elucidated, but its regulatory mechanism remains largely unknown. In this report, we biochemically identified a CCAAT-box transcription factor (SlNFYA10) that can bind to the promoter of SlGME1, which encodes GDP-Man-3’,5’-epimerase, a pivotal enzyme in the d-mannose/l-galactose pathway. Importantly, SlNFYA10 simultaneously binds to the promoter of SlGGP1, a downstream gene of SlGME1 in the d-mannose/l-galactose pathway. Binding assays in yeast and functional analyses in plants have confirmed that SlNFYA10 exerts a negative effect on the expression of both SlGME1 and SlGGP1. Transgenic tomato lines overexpressing SlNFYA10 show decreased levels of SlGME1 and SlGGP1 abundance and AsA concentration in their leaves and fruits, accompanied by enhanced sensitivity to oxidative stress. Overall, SlNFYA10 is the first CCAAT-binding factor identified to date to negatively regulate the AsA biosynthetic pathway at multiple sites and modulate plant responses to oxidative stress.