Angus M. MacNicol

Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF

Animal cap explants from Xenopus embryos injected with a dominant negative Raf-1 mutant, termed NAF (not a functional Raf), demonstrated a complete block to basic fibroblast growth factor (FGF)-stimulated mesoderm induction. Activin induction of mesoderm was normal in embryos that expressed NAF. Injection of NAF RNA into 2-cell stage embryos blocked normal development during neurula stages and caused severe posterior truncations in tadpoles. The phenotype induced by NAF could be rescued by coinjection of wild-type raf-1 RNA. The NAF mutant functioned by specifically blocking the activation of endogenous Raf kinase activity. These findings suggest that Raf-1 mediates FGF, but not activin, receptor signaling during mesoderm induction and implicate Raf-1 as a key signaling molecule in the development of posterior structure.

Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation

A strict temporal order of maternal mRNA translation is essential for meiotic cell cycle progression in oocytes of the frog Xenopus laevis. The molecular mechanisms controlling the ordered pattern of mRNA translational activation have not been elucidated. We report a novel role for the neural stem cell regulatory protein, Musashi, in controlling the translational activation of the mRNA encoding the Mos proto‐oncogene during meiotic cell cycle progression. We demonstrate that Musashi interacts specifically with the polyadenylation response element in the 3′ untranslated region of the Mos mRNA and that this interaction is necessary for early Mos mRNA translational activation. A dominant inhibitory form of Musashi blocks maternal mRNA cytoplasmic polyadenylation and meiotic cell cycle progression. Our data suggest that Musashi is a target of the initiating progesterone signaling pathway and reveal that late cytoplasmic polyadenylation element‐directed mRNA translation requires early, Musashi‐dependent mRNA translation. These findings indicate that Musashi function is necessary to establish the temporal order of maternal mRNA translation during Xenopus meiotic cell cycle progression.

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.

The mitogen-activated protein kinase signaling pathway stimulates mos mRNA cytoplasmic polyadenylation during Xenopus oocyte maturation.

ABSTRACT The Mos protein kinase is a key regulator of vertebrate oocyte maturation. Oocyte-specific Mos protein expression is subject to translational control. In the frog Xenopus, the translation of Mos protein requires the progesterone-induced polyadenylation of the maternal Mos mRNA, which is present in the oocyte cytoplasm. Both theXenopus p42 mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) signaling pathways have been proposed to mediate progesterone-stimulated oocyte maturation. In this study, we have determined the relative contributions of the MAPK and MPF signaling pathways to Mos mRNA polyadenylation. We report that progesterone-induced Mos mRNA polyadenylation was attenuated in oocytes expressing the MAPK phosphatase rVH6. Moreover, inhibition of MAPK signaling blocked progesterone-induced Mos protein accumulation. Activation of the MAPK pathway by injection of RNA encoding Mos was sufficient to induce both the polyadenylation of synthetic Mos mRNA substrates and the accumulation of endogenous Mos protein in the absence of MPF signaling. Activation of MPF, by injection of cyclin B1 RNA or purified cyclin B1 protein, also induced both Mos protein accumulation and Mos mRNA polyadenylation. However, this action of MPF required MAPK activity. By contrast, the cytoplasmic polyadenylation of maternal cyclin B1 mRNA was stimulated by MPF in a MAPK-independent manner, thus revealing a differential regulation of maternal mRNA polyadenylation by the MAPK and MPF signaling pathways. We propose that MAPK-stimulated Mos mRNA cytoplasmic polyadenylation is a key component of the positive-feedback loop, which contributes to the all-or-none process of oocyte maturation.

A novel regulatory element determines the timing of Mos mRNA translation during Xenopus oocyte maturation

Progression through vertebrate oocyte maturation requires that pre‐existing, maternally derived mRNAs be translated in a strict temporal order. The mechanism that controls the timing of oocyte mRNA translation is unknown. In this study we show that the early translational induction of the mRNA encoding the Mos proto‐oncogene is mediated through a novel regulatory element within the 3′ untranslated region of the Mos mRNA. This novel element is responsive to the MAP kinase signaling pathway and is distinct from the late acting, cdc2‐responsive, cytoplasmic polyadenylation element. Our findings suggest that the timing of maternal mRNA translation is controlled through signal transduction pathways targeting distinct 3′ UTR mRNA elements.

Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system.

BACKGROUND The expression patterns of the segment polarity genes wingless and engrailed are conserved during segmentation in a variety of arthropods, suggesting that the regulatory interactions between these two genes are also evolutionarily conserved. Hypotheses derived from such comparisons of gene expression patterns are difficult to test experimentally as genetic manipulation is currently possible for only a few model organisms. RESULTS We have developed a system, using recombinant baculoviruses, that can be applied to a wide variety of organisms to study the effects of ectopic expression of genes. As a first step, we studied the range and type of infection of several reporter viruses in the embryos of two arthropod and one vertebrate species. Using this system to express wingless, we were able to induce expression of engrailed in the anterior half of each parasegment in embryos of the fruit fly Drosophila melanogaster. Virus-mediated wingless expression also caused ectopic naked ventral cuticle formation in wild-type Drosophila larvae. In the flour beetle, Tribolium castaneum, ectopic wingless also induced engrailed expression. As in Drosophila, this expression was only detectable in the anterior half of the parasegment. CONCLUSIONS The functional interaction between wingless and engrailed, and the establishment of cells competent to express engrailed, appears to be conserved between Drosophila and Tribolium. The data on the establishment of an engrailed-competent domain also support the idea that prepatterning by pair-rule genes is conserved between these two insects. The recombinant baculovirus technology reported here may help answer other long-standing comparative evolutionary questions.

Enforcing temporal control of maternal mRNA translation during oocyte cell-cycle progression

Meiotic cell‐cycle progression in progesterone‐stimulated Xenopus oocytes requires that the translation of pre‐existing maternal mRNAs occur in a strict temporal order. Timing of translation is regulated through elements within the mRNA 3′ untranslated region (3′ UTR), which respond to cell cycle‐dependant signalling. One element that has been previously implicated in the temporal control of mRNA translation is the cytoplasmic polyadenylation element (CPE). In this study, we show that the CPE does not direct early mRNA translation. Rather, early translation is directed through specific early factors, including the Musashi‐binding element (MBE) and the MBE‐binding protein, Musashi. Our findings indicate that although the cyclin B5 3′ UTR contains both CPEs and an MBE, the MBE is the critical regulator of early translation. The cyclin B2 3′ UTR contains CPEs, but lacks an MBE and is translationally activated late in maturation. Finally, utilizing antisense oligonucleotides to attenuate endogenous Musashi synthesis, we show that Musashi is critical for the initiation of early class mRNA translation and for the subsequent activation of CPE‐dependant mRNA translation.

Context-dependent regulation of Musashi-mediated mRNA translation and cell cycle regulation.

Musashi-mediated mRNA translational control has been implicated in the promotion of physiological and pathological stem cell proliferation. During self-renewal of mammalian stem cells, Musashi has been proposed to act to repress the translation of mRNAs encoding inhibitors of cell cycle progression. By contrast, in maturing Xenopus oocytes Musashi activates translation of target mRNAs that encode proteins promoting cell cycle progression. The mechanisms directing Musashi to differentially control mRNA translation in mammalian stem cells and Xenopus oocytes is unknown. In this study, we demonstrate that the mechanisms defining Musashi function lie within the cellular context. Specifically, we show that murine Musashi acts as an activator of translation in maturing Xenopus oocytes while Xenopus Musashi functions as a repressor of target mRNA translation in mammalian cells. We further demonstrate that within the context of a primary mammalian neural stem/progenitor cell, Musashi can be converted from a repressor of mRNA translation to an activator of translation in response to extracellular stimuli. We present current models of Musashi-mediated mRNA translational control and discuss possible mechanisms for regulating Musashi function. An understanding of these mechanisms presents exciting possibilities for development of therapeutic targets to control physiological and pathological stem cell proliferation.

Disruption of the 14-3-3 Binding Site within the B-Raf Kinase Domain Uncouples Catalytic Activity from PC12 Cell Differentiation

A number of Raf-associated proteins have recently been identified, including members of the 14-3-3 family of phosphoserine-binding proteins. Although both positive and negative regulatory functions have been ascribed for 14-3-3 interactions with Raf-1, the mechanisms by which 14-3-3 binding modulates Raf activity have not been fully established. We report that mutational disruption of 14-3-3 binding to the B-Raf catalytic domain inhibits B-Raf biological activity. Expression of the isolated B-Raf catalytic domain (B-Rafcat) induces PC12 cell differentiation in the absence of nerve growth factor. By contrast, the B-Rafcat 14-3-3 binding mutant, B-Rafcat S728A, was severely compromised for the induction of PC12 cell differentiation. Interestingly, the B-Rafcat 14-3-3 binding mutant retained significant in vitro catalytic activity. InXenopus oocytes, the analogous full-length B-Raf 14-3-3 binding mutant blocked progesterone-stimulated maturation and the activation of endogenous mitogen-activated protein kinase kinase and mitogen-activated protein kinase. Similarly, the full-length B-Raf 14-3-3 binding mutant inhibited nerve growth factor-stimulated PC12 cell differentiation. We conclude that 14-3-3 interaction with the catalytic domain is not required for kinase activity per sebut is essential to couple B-Raf catalytic activity to downstream effector activation.

Function and regulation of the mammalian Musashi mRNA translational regulator.

The evolutionarily conserved RNA-binding protein, Musashi, regulates neural stem cell self-renewal. Musashi expression is also indicative of stem cell populations in breast and intestinal tissues and is linked to cell overproliferation in cancers of these tissues. Musashi has been primarily implicated as a repressor of target mRNAs in stem cell populations. However, little is known about the mechanism by which Musashi exerts mRNA translational control or how Musashi function is regulated. Recent findings in oocytes of the frog, Xenopus, indicate an unexpected role for Musashi as an activator of a number of maternal mRNAs during meiotic cell cycle progression. Given the importance of Musashi function in stem cell biology and the implications of aberrant Musashi expression in cancer, it is critical that we understand the molecular processes that regulate Musashi function.