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The anterior pituitary controls key biological processes, including growth, metabolism, reproduction, and stress responses through distinct cell types that each secrete specific hormones. The anterior pituitary cells show a remarkable level of cell type plasticity that mediates the shifts in hormone-producing cell populations that are required to meet organismal needs. The molecular mechanisms underlying pituitary cell plasticity are not well understood. Recent work has implicated the pituitary stem cell populations and specifically, the mRNA binding proteins of the Musashi family in control of pituitary cell type identity. In this study we have identified the target mRNAs that mediate Musashi function in the adult mouse pituitary and demonstrate the requirement for Musashi function in vivo. Using Musashi RNA immunoprecipitation, we identify a cohort of 1184 mRNAs that show specific Musashi binding. Identified Musashi targets include the Gnrhr mRNA, which encodes the gonadotropin-releasing hormone receptor (GnRHR), and the Fshb mRNA, encoding follicle-stimulating hormone (FSH). Reporter assays reveal that Musashi functions to exert repression of translation of the Fshb mRNA, in addition to the previously observed repression of the Gnrhr mRNA. Importantly, mice engineered to lack Musashi in gonadotropes demonstrate a failure to repress translation of the endogenous Gnrhr and Fshb mRNAs during the estrous cycle and display a significant heterogeneity in litter sizes. The range of identified target mRNAs suggests that, in addition to these key gonadotrope proteins, Musashi may exert broad regulatory control over the pituitary proteome in a cell type–specific manner. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.02.033 Byline: Yi Ying Wang (a), Amanda Charlesworth (b)(c), Shannon M. Byrd (e), Robert Gregerson (f), Melanie C. MacNicol (b)(c), Angus M. MacNicol (a)(b)(d) Abstract: Cell cycle progression during oocyte maturation requires the strict temporal regulation of maternal mRNA translation. The intrinsic basis of this temporal control has not been fully elucidated but appears to involve distinct mRNA 3' UTR regulatory elements. In this study, we identify a novel translational control sequence (TCS) that exerts repression of target mRNAs in immature oocytes of the frog, Xenopus laevis, and can direct early cytoplasmic polyadenylation and translational activation during oocyte maturation. The TCS is functionally distinct from the previously characterized Musashi/polyadenylation response element (PRE) and the cytoplasmic polyadenylation element (CPE). We report that TCS elements exert translational repression in both the Wee1 mRNA 3' UTR and the pericentriolar material-1 (Pcm-1) mRNA 3' UTR in immature oocytes. During oocyte maturation, TCS function directs the early translational activation of the Pcm-1 mRNA. By contrast, we demonstrate that CPE sequences flanking the TCS elements in the Wee1 3' UTR suppress the ability of the TCS to direct early translational activation. Our results indicate that a functional hierarchy exists between these distinct 3' UTR regulatory elements to control the timing of maternal mRNA translational activation during oocyte maturation. Author Affiliation: (a) Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Slot 814, 4301 W. Markham St., Little Rock, AR 72205, USA (b) Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Slot 814, 4301 W. Markham St., Little Rock, AR 72205, USA (c) The Center for Translational Neuroscience, University of Arkansas for Medical Sciences, Slot 814, 4301 W. Markham St., Little Rock, AR 72205, USA (d) The Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Slot 814, 4301 W. Markham St., Little Rock, AR 72205, USA (e) Committee on Developmental Biology, The University of Chicago, Chicago, IL, USA (f) Science Division, Lyon College, Batesville, AR, USA Article History: Received 5 June 2007; Revised 14 February 2008; Accepted 15 February 2008

A healthy nutritional state is required for all aspects of reproduction and is signaled by the adipokine leptin. Leptin acts in a relatively narrow concentration range: too much or too little will compromise fertility. The leptin signal timing is important to prepubertal development in both sexes. In the brain, leptin acts on ventral premammillary neurons which signal kisspeptin (Kiss1) neurons to stimulate gonadotropin releasing hormone (GnRH) neurons. Suppression of Kiss1 neurons occurs when agouti-related peptide neurons are activated by reduced leptin, because leptin normally suppresses these orexigenic neurons. In the pituitary, leptin stimulates production of GnRH receptors (GnRHRs) and follicle-stimulating hormone at midcycle, by activating pathways that derepress actions of the messenger ribonucleic acid translational regulatory protein Musashi. In females, rising estrogen stimulates a rise in serum leptin, which peaks at midcycle, synchronizing with nocturnal luteinizing hormone pulses. The normal range of serum leptin levels (10-20 ng/mL) along with gonadotropins and growth factors promote ovarian granulosa and theca cell functions and oocyte maturation. In males, the prepubertal rise in leptin promotes testicular development. However, a decline in leptin levels in prepubertal boys reflects inhibition of leptin secretion by rising androgens. In adult males, leptin levels are 10% to 50% of those in females, and high leptin inhibits testicular function. The obesity epidemic has elucidated leptin resistance pathways, with too much leptin in either sex leading to infertility. Under conditions of balanced nutrition, however, the secretion of leptin is timed and regulated within a narrow level range that optimizes its trophic effects. [ABSTRACT FROM AUTHOR]

Anterior pituitary somatotropes are important metabolic sensors responding to leptin by secreting growth hormone (GH). However, reduced leptin signals caused by fasting have not always correlated with reduced serum GH. Reports show that fasting may stimulate or reduce GH secretion, depending on the species. Mechanisms underlying these distinct somatotrope responses to fasting remain unknown. To define the s omatotrope response to decreased leptin signaling we examined markers of somatotrope function over different time periods of fasting. Male mice were fasted for 24 and 48 h, with female mice fasted for 24 h compared to fed controls ad libitum. Body weight and serum glucose were reduced in both males and females, but, unexpectedly, serum leptin was reduced only in males. Furthermore, in males, serum GH levels showed a biphasic response with significant reductions at 24 h followed by a significant rise at 48 h, which coincided with the rise in serum ghrelin levels. In contrast, females showed an increase in serum GH at 24 h. We then explored mechanisms underlying the differential so matotrope responses seen in males and observed that pituitary levels of Gh mRNA increased, with no distinction between acute and prolonged fasting. By contrast, the Ghrhr mRNA (encoding GH releasing hormone receptor) and the Ghsr mRNA (encoding the ghrelin receptor) were both greatly increased at prolonged fasting times coincident with increased serum GH. These findings show sex differences in the somatotrope and ad ipocyte responses to fasting and support an adaptive role for somatotropes in males in response to multiple metabolic signals. [ABSTRACT FROM AUTHOR]

The Musashi family of mRNA translational regulators controls both physiological and pathological stem cell self-renewal primarily by repressing target mRNAs that promote differentiation. In response to differentiation cues, Musashi can switch from a repressor to an activator of target mRNA translation. However, the molecular events that distinguish Musashi-mediated translational activation from repression are not understood. We have previously reported that Musashi function is required for the maturation of Xenopus oocytes and specifically for translational activation of specific dormant maternal mRNAs. Here, we employed MS to identify cellular factors necessary for Musashi-dependent mRNA translational activation. We report that Musashi1 needs to associate with the embryonic poly(A)-binding protein (ePABP) or the canonical somatic cell poly(A)-binding protein PABPC1 for activation of Musashi target mRNA translation. Co-immunoprecipitation studies demonstrated an increased Musashi1 interaction with ePABP during oocyte maturation. Attenuation of endogenous ePABP activity severely compromised Musashi function, preventing downstream signaling and blocking oocyte maturation. Ectopic expression of either ePABP or PABPC1 restored Musashi-dependent mRNA translational activation and maturation of ePABP-attenuated oocytes. Consistent with these Xenopus findings, PABPC1 remained associated with Musashi under conditions of Musashi target mRNA de-repression and translation during mammalian stem cell differentiation. Because association of Musashi1 with poly(A)-binding proteins has previously been implicated only in repression of Musashi target mRNAs, our findings reveal novel context-dependent roles for the interaction of Musashi with poly(A)-binding protein family members in response to extracellular cues that control cell fate. [ABSTRACT FROM AUTHOR]

Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease. [ABSTRACT FROM AUTHOR]

Background: There is increasing evidence of a pivotal role for regulated mRNA translation in control of developmental cell fate transitions. Physiological and pathological stem and progenitor cell self-renewal is maintained by the mRNA-binding protein, Musashi1 through repression of translation of key mRNAs encoding cell cycle inhibitory proteins. The mechanism by which Musashi1 function is modified to allow translation of these target mRNAs under conditions that require inhibition of cell cycle progression, is unknown. Results: In this study, we demonstrate that differentiation of primary embryonic rat neural stem/progenitor cells (NSPCs) or human neuroblastoma SH-SY5Y cells results in the rapid phosphorylation of Musashi1 on the evolutionarily conserved site serine 337 (S337). Phosphorylation of this site has been shown to be required for cell cycle control during the maturation of Xenopus oocytes. S337 phosphorylation in mammalian NSPCs and human SH-SY5Y cells correlates with the de-repression and translation of a Musashi reporter mRNA and with accumulation of protein from the endogenous Musashi target mRNA, p21WAF1/CIP1. Inhibition of Musashi regulatory phosphorylation, through expression of a phospho-inhibitory mutant Musashi1 S337A or over-expression of the wild-type Musashi, blocked differentiation of both NSPCs and SH-SY5Y cells. Musashi1 was similarly phosphorylated in NSPCs and SH-SY5Y cells under conditions of nutrient deprivation-induced cell cycle arrest. Expression of the Musashi1 S337A mutant protein attenuated nutrient deprivation-induced NSPC and SH-SY5Y cell death. Conclusions: Our data suggest that in response to environmental cues that oppose cell cycle progression, regulation of Musashi function is required to promote target mRNA translation and cell fate transition. Forced modulation of Musashi1 function may present a novel therapeutic strategy to oppose pathological stem cell self-renewal. [ABSTRACT FROM AUTHOR]

Cell cycle re-entry during vertebrate oocyte maturation is mediated through translational activation of select target mRNAs, culminating in the activation of mitogen-activated protein kinase and cyclin B/cyclin-dependent kinase (CDK) signaling. The temporal order of targeted mRNA translation is crucial for cell cycle progression and is determined by the timing of activation of distinct mRNA-binding proteins. We have previously shown in oocytes from Xenopus laevis that the mRNA-binding protein Musashi targets translational activation of early class mRNAs including the mRNA encoding the Mos proto-oncogene. However, the molecular mechanism by which Musashi function is activated is unknown. We report here that activation of Musashi1 is mediated by Ringo/CDK signaling, revealing a novel role for early Ringo/CDK function. Interestingly, Musashi1 activation is subsequently sustained through mitogen-activated protein kinase signaling, the downstream effector of Mos mRNA translation, thus establishing a positive feedback loop to amplify Musashi function. The identified regulatory sites are present in mammalian Musashi proteins, and our data suggest that phosphorylation may represent an evolutionarily conserved mechanism to control Musashi-dependent target mRNA translation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]