The Musashi RNA binding proteins direct the translational activation of key pituitary mRNAs.

The pituitary functions as a master endocrine gland that secretes hormones critical for regulation of a wide variety of physiological processes including reproduction, growth, metabolism and stress responses. The distinct hormone-producing cell lineages within the pituitary display remarkable levels of cell plasticity that allow remodeling of the relative proportions of each hormone-producing cell population to meet organismal demands. The molecular mechanisms governing pituitary cell plasticity have not been fully elucidated. Our recent studies have implicated a role for the Musashi family of sequence-specific mRNA binding proteins in the control of pituitary hormone production, pituitary responses to hypothalamic stimulation and modulation of pituitary transcription factor expression in response to leptin signaling. To date, these actions of Musashi in the pituitary appear to be mediated through translational repression of the target mRNAs. Here, we report Musashi1 directs the translational activation, rather than repression, of the Prop1, Gata2 and Nr5a1 mRNAs which encode key pituitary lineage specification factors. We observe that Musashi1 further directs the translational activation of the mRNA encoding the glycolipid Neuronatin (Nnat) as determined both in mRNA reporter assays as well as in vivo. Our findings suggest a complex bifunctional role for Musashi1 in the control of pituitary cell function.

Anterior Pituitary Transcriptomics Following a High-Fat Diet: Impact of Oxidative Stress on Cell Metabolism.

Anterior pituitary cell function requires a high level of protein synthesis and secretion which depend heavily on mitochondrial adenosine triphosphate production and functional endoplasmic reticula. Obesity adds stress to tissues, requiring them to adapt to inflammation and oxidative stress, and adding to their allostatic load. We hypothesized that pituitary function is vulnerable to the stress of obesity. Here, we utilized a 10- to 15-week high-fat diet (HFD, 60%) in a thermoneutral environment to promote obesity, testing both male and female FVB.129P mice. We quantified serum hormones and cytokines, characterized the metabolic phenotype, and defined changes in the pituitary transcriptome using single-cell RNA-sequencing analysis. Weight gain was significant by 3 weeks in HFD mice, and by 10 weeks all HFD groups had gained 20 g. HFD females (15 weeks) had increased energy expenditure and decreased activity. All HFD groups showed increases in serum leptin and decreases in adiponectin. HFD caused increased inflammatory markers: interleukin-6, resistin, monocyte chemoattractant protein-1, and tumor necrosis factorα. HFD males and females also had increased insulin and increased TSH, and HFD females had decreased serum prolactin and growth hormone pulse amplitude. Pituitary single-cell transcriptomics revealed modest or no changes in pituitary cell gene expression from HFD males after 10 or 15 weeks or from HFD females after 10 weeks. However, HFD females (15 weeks) showed significant numbers of differentially expressed genes in lactotropes and pituitary stem cells. Collectively, these studies reveal that pituitary cells from males appear to be more resilient to the oxidative stress of obesity than females and identify the most vulnerable pituitary cell populations in females.

Musashi Exerts Control of Gonadotrope Target mRNA Translation During the Mouse Estrous Cycle.

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.

Anti-glioblastoma activity of monensin and its analogs in an organoid model of cancer.

Glioblastoma (GBM) remains the most frequently diagnosed primary malignant brain cancer in adults. Despite recent progress in understanding the biology of GBM, the clinical outcome for patients remains poor, with a median survival of approximately one year after diagnosis. One factor contributing to failure in clinical trials is the fact that traditional models used in GBM drug discovery poorly recapitulate patient tumors. Previous studies have shown that monensin (MON) analogs, namely esters and amides on C-26 were potent towards various types of cancer cell lines. In the present study we have investigated the activity of these molecules in GBM organoids, as well as in a host:tumor organoid model. Using a mini-ring cell viability assay we have identified seven analogs (IC50 = 91.5 ± 54.4-291.7 ± 68.8 nM) more potent than parent MON (IC50 = 612.6 ± 184.4 nM). Five of these compounds induced substantial DNA fragmentation in GBM organoids, suggestive of apoptotic cell death. The most active analog, compound 1, significantly reduced GBM cell migration, induced PARP degradation, diminished phosphorylation of STAT3, Akt and GSK3ß, increased É£H2AX signaling and upregulated expression of the autophagy associated marker LC3-II. To investigate the activity of MON and compound 1 in a tumor microenvironment, we developed human cerebral organoids (COs) from human induced pluripotent stem cells (iPSCs). The COs showed features of early developing brain such as multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1 +), primitive ventricular system (SOX2 +/Ki67 +), intermediate zone (TBR2 +) and cortical plate (MAP2 +). In order to generate host:tumor organoids, we co-cultured RFP-labeled U87MG cells with fully formed COs. Compound 1 and MON reduced U87MG tumor size in the COs after four days of treatment and induced a significant reduction of PARP expression. These findings highlight the therapeutic potential of MON analogs towards GBM and support the application of organoid models in anti-cancer drug discovery.

Single and double modified salinomycin analogs target stem-like cells in 2D and 3D breast cancer models.

Breast cancer remains one of the leading cancers among women. Cancer stem cells (CSCs) are tumor-initiating cells which drive progression, metastasis, and reoccurrence of the disease. CSCs are resistant to conventional chemo- and radio-therapies and their ability to survive such treatment enables tumor reestablishment. Metastasis is the main cause of mortality in women with breast cancer, thus advances in treatment will depend on therapeutic strategies targeting CSCs. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic known for its anticancer activity towards several types of tumor cells. In the present work, a library of 17 C1-single and C1/C20-double modified SAL analogs was screened to identify compounds with improved activity against breast CSCs. Six single- and two double-modified analogs were more potent (IC50 range of 1.1 ± 0.1-1.4 ± 0.2 µM) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC50 of 4.9 ± 1.6 µM). Double-modified compound 17 was found to be more efficacious than SAL against the majority of cancer cell lines in the NCI-60 Human Tumor Cell Line Panel. Compound 17 was more potent than SAL in inhibiting cell migration and cell renewal properties of MDA-MB-231 cells, as well as inducing selective loss of the CD44+/CD24/low stem-cell-like subpopulation in both monolayer (2D) and organoid (3D) culture. The present findings highlight the therapeutic potential of SAL analogs towards breast CSCs and identify select compounds that merit further study and clinical development.

Post-Transcriptional Regulation of Gnrhr: A Checkpoint for Metabolic Control of Female Reproduction.

The proper expression of gonadotropin-releasing hormone receptors (GnRHRs) by pituitary gonadotropes is critical for maintaining maximum reproductive capacity. GnRH receptor expression must be tightly regulated in order to maintain the normal pattern of expression through the estrous cycle in rodents, which is believed to be important for interpreting the finely tuned pulses of GnRH from the hypothalamus. Much work has shown that Gnrhr expression is heavily regulated at the level of transcription. However, researchers have also discovered that Gnrhr is regulated post-transcriptionally. This review will discuss how RNA-binding proteins and microRNAs may play critical roles in the regulation of GnRHR expression. We will also discuss how these post-transcriptional regulators may themselves be affected by metabolic cues, specifically with regards to the adipokine leptin. All together, we present evidence that Gnrhr is regulated post-transcriptionally, and that this concept must be further explored in order to fully understand the complex nature of this receptor.

The Importance of Leptin to Reproduction.

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.

Control of the Anterior Pituitary Cell Lineage Regulator POU1F1 by the Stem Cell Determinant Musashi.

The adipokine leptin regulates energy homeostasis through ubiquitously expressed leptin receptors. Leptin has a number of major signaling targets in the brain, including cells of the anterior pituitary (AP). We have previously reported that mice lacking leptin receptors in AP somatotropes display growth hormone (GH) deficiency, metabolic dysfunction, and adult-onset obesity. Among other targets, leptin signaling promotes increased levels of the pituitary transcription factor POU1F1, which in turn regulates the specification of somatotrope, lactotrope, and thyrotrope cell lineages within the AP. Leptin's mechanism of action on somatotropes is sex dependent, with females demonstrating posttranscriptional control of Pou1f1 messenger RNA (mRNA) translation. Here, we report that the stem cell marker and mRNA translational control protein, Musashi1, exerts repression of the Pou1f1 mRNA. In female somatotropes, Msi1 mRNA and protein levels are increased in the mouse model that lacks leptin signaling (Gh-CRE Lepr-null), coincident with lack of POU1f1 protein, despite normal levels of Pou1f1 mRNA. Single-cell RNA sequencing of pituitary cells from control female animals indicates that both Msi1 and Pou1f1 mRNAs are expressed in Gh-expressing somatotropes, and immunocytochemistry confirms that Musashi1 protein is present in the somatotrope cell population. We demonstrate that Musashi interacts directly with the Pou1f1 mRNA 3' untranslated region and exerts translational repression of a Pou1f1 mRNA translation reporter in a leptin-sensitive manner. Musashi immunoprecipitation from whole pituitary reveals coassociated Pou1f1 mRNA. These findings suggest a mechanism in which leptin stimulation is required to reverse Musashi-mediated Pou1f1 mRNA translational control to coordinate AP somatotrope function with metabolic status.

Sex differences in somatotrope response to fasting: biphasic responses in male mice.

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 somatotrope 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 somatotrope 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 adipocyte responses to fasting and support an adaptive role for somatotropes in males in response to multiple metabolic signals.

Molecular Mechanisms of Pituitary Cell Plasticity.

The mechanisms that mediate plasticity in pituitary function have long been a subject of vigorous investigation. Early studies overcame technical barriers and challenged conceptual barriers to identify multipotential and multihormonal cell populations that contribute to diverse pituitary stress responses. Decades of intensive study have challenged the standard model of dedicated, cell type-specific hormone production and have revealed the malleable cellular fates that mediate pituitary responses. Ongoing studies at all levels, from animal physiology to molecular analyses, are identifying the mechanisms underlying this cellular plasticity. This review describes the findings from these studies that utilized state-of-the-art tools and techniques to identify mechanisms of plasticity throughout the pituitary and focuses on the insights brought to our understanding of pituitary function.

Metabolic signalling to somatotrophs: Transcriptional and post-transcriptional mediators.

In normal individuals, pituitary somatotrophs optimise body composition by responding to metabolic signals from leptin. To identify mechanisms behind the regulation of somatotrophs by leptin, we used Cre-LoxP technology to delete leptin receptors (LEPR) selectively in somatotrophs and developed populations purified by fluorescence-activated cell sorting (FACS) that contained 99% somatotrophs. FACS-purified, Lepr-null somatotrophs showed reduced levels of growth hormone (GH), growth hormone-releasing hormone receptor (GHRHR), and Pou1f1 proteins and Gh (females) and Ghrhr (both sexes) mRNAs. Pure somatotrophs also expressed thyroid-stimulating hormone (TSH) and prolactin (PRL), both of which were reduced in pure somatotrophs lacking LEPR. This introduced five gene products that were targets of leptin. In the present study, we tested the hypothesis that leptin is both a transcriptional and a post-transcriptional regulator of these gene products. Our tests showed that Pou1f1 and/or the Janus kinase/signal transducer and activator of transcription 3 transcriptional regulatory pathways are implicated in the leptin regulation of Gh or Ghrhr mRNAs. We then focused on potential actions by candidate microRNAs (miRNAs) with consensus binding sites on the 3' UTR of Gh or Ghrhr mRNAs. Somatotroph Lepr-null deletion mutants expressed elevated levels of miRNAs including miR1197-3p (in females), miR103-3p and miR590-3p (both sexes), which bind Gh mRNA, or miRNA-325-3p (elevated in both sexes), which binds Ghrhr mRNA. This elevation indicates repression of translation in the absence of LEPR. In addition, after detecting binding sites for Musashi on Tshb and Prl 3' UTR, we determined that Musashi1 repressed translation of both mRNAs in in vitro fluc assays and that Prl mRNA was enriched in Musashi immunoprecipitation assays. Finally, we tested ghrelin actions to determine whether its nitric oxide-mediated signalling pathways would restore somatotroph functions in deletion mutants. Ghrelin did not restore either GHRH binding or GH secretion in vitro. These studies show an unexpectedly broad role for leptin with respect to maintaining somatotroph functions, including the regulation of PRL and TSH in subsets of somatotrophs that may be progenitor cells.

Musashi interaction with poly(A)-binding protein is required for activation of target mRNA translation.

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.

Association of Gnrhr mRNA With the Stem Cell Determinant Musashi: A Mechanism for Leptin-Mediated Modulation of GnRHR Expression.

The cyclic expression of pituitary gonadotropin-releasing hormone receptors (GnRHRs) may be an important checkpoint for leptin regulatory signals. Gonadotrope Lepr-null mice have reduced GnRHR levels, suggesting these receptors may be leptin targets. To determine if leptin stimulated GnRHR directly, primary pituitary cultures or pieces were exposed to 1 to 100 nM leptin. Leptin increased GnRHR protein levels and the percentages of gonadotropes that bound biotinylated analogs of gonadotropin-releasing hormone (bio-GnRH) but had no effect on Gnrhr messenger RNA (mRNA). An in silico analysis revealed three consensus Musashi (MSI) binding elements (MBEs) for this translational control protein in the 3' untranslated region (UTR) of Gnrhr mRNA. Several experiments determined that these Gnrhr mRNA MBE were active: (1) RNA electrophoretic mobility shift assay analyses showed that MSI1 specifically bound Gnrhr mRNA 3'-UTR; (2) RNA immunoprecipitation of pituitary fractions with MSI1 antibody pulled down a complex enriched in endogenous MSI protein and endogenous Gnrhr mRNA; and (3) fluorescence reporter assays showed that MSI1 repressed translation of the reporter coupled to the Gnrhr 3'-UTR. In vitro, leptin stimulation of pituitary pieces reduced Msi1 mRNA in female pituitaries, and leptin stimulation of pituitary cultures reduced MSI1 proteins selectively in gonadotropes identified by binding to bio-GnRH. These findings show that leptin's direct stimulatory actions on gonadotrope GnRHR correlate with a direct inhibition of expression of the posttranscriptional regulator MSI1. We also show MSI1 interaction with the 3'-UTR of Gnrhr mRNA. These findings now open the door to future studies of leptin-modulated posttranscriptional pathways.

Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2.

The Musashi family of RNA binding proteins act to promote stem cell self-renewal and oppose cell differentiation predominantly through translational repression of mRNAs encoding pro-differentiation factors and inhibitors of cell cycle progression. During tissue development and repair however, Musashi repressor function must be dynamically regulated to allow cell cycle exit and differentiation. The mechanism by which Musashi repressor function is attenuated has not been fully established. Our prior work indicated that the Musashi1 isoform undergoes site-specific regulatory phosphorylation. Here, we demonstrate that the canonical Musashi2 isoform is subject to similar regulated site-specific phosphorylation, converting Musashi2 from a repressor to an activator of target mRNA translation. We have also characterized a novel alternatively spliced, truncated isoform of human Musashi2 (variant 2) that lacks the sites of regulatory phosphorylation and fails to promote translation of target mRNAs. Consistent with a role in opposing cell cycle exit and differentiation, upregulation of Musashi2 variant 2 was observed in a number of cancers and overexpression of the Musashi2 variant 2 isoform promoted cell transformation. These findings indicate that alternately spliced isoforms of the Musashi protein family possess distinct functional and regulatory properties and suggest that differential expression of Musashi isoforms may influence cell fate decisions.

Leptin Regulation of Gonadotrope Gonadotropin-Releasing Hormone Receptors As a Metabolic Checkpoint and Gateway to Reproductive Competence.

The adipokine leptin signals the body's nutritional status to the brain, and particularly, the hypothalamus. However, leptin receptors (LEPRs) can be found all throughout the body and brain, including the pituitary. It is known that leptin is permissive for reproduction, and mice that cannot produce leptin (Lep/Lep) are infertile. Many studies have pinpointed leptin's regulation of reproduction to the hypothalamus. However, LEPRs exist at all levels of the hypothalamic-pituitary-gonadal axis. We have previously shown that deleting the signaling portion of the LEPR specifically in gonadotropes impairs fertility in female mice. Our recent studies have targeted this regulation to the control of gonadotropin releasing hormone receptor (GnRHR) expression. The hypotheses presented here are twofold: (1) cyclic regulation of pituitary GnRHR levels sets up a target metabolic checkpoint for control of the reproductive axis and (2) multiple checkpoints are required for the metabolic signaling that regulates the reproductive axis. Here, we emphasize and explore the relationship between the hypothalamus and the pituitary with regard to the regulation of GnRHR. The original data we present strengthen these hypotheses and build on our previous studies. We show that we can cause infertility in 70% of female mice by deleting all isoforms of LEPR specifically in gonadotropes. Our findings implicate activin subunit (InhBa) mRNA as a potential leptin target in gonadotropes. We further show gonadotrope-specific upregulation of GnRHR protein (but not mRNA levels) following leptin stimulation. In order to try and understand this post-transcriptional regulation, we tested candidate miRNAs (identified with in silico analysis) that may be binding the Gnrhr mRNA. We show significant upregulation of one of these miRNAs in our gonadotrope-Lepr-null females. The evidence provided here, combined with our previous work, lay the foundation for metabolically regulated post-transcriptional control of the gonadotrope. We discuss possible mechanisms, including miRNA regulation and the involvement of the RNA binding protein, Musashi. We also demonstrate how this regulation may be vital for the dynamic remodeling of gonadotropes in the cycling female. Finally, we propose that the leptin receptivity of both the hypothalamus and the pituitary are vital for the body's ability to delay or slow reproduction during periods of low nutrition.

A Sex-Dependent, Tropic Role for Leptin in the Somatotrope as a Regulator of POU1F1 and POU1F1-Dependent Hormones.

Pituitary somatotropes perform the key function of coordinating organismic growth and body composition with metabolic signals. However, the mechanism by which they sense and respond to metabolic signals via the adipokine leptin is unknown. The complex interplay between the heterogeneous cell types of the pituitary confounds the identification of somatotrope-specific mechanisms. Somatotropes represent 30%-40% of the anterior pituitary population and are derived from a lineage of cells that are activated by the Pit-Oct-Unc domain family domain class 1 transcription factor 1 (POU1F1) to produce GH, prolactin (PRL). and TSH. To determine the mechanism by which leptin controls somatotrope function, we used Cre-LoxP technology and fluorescence-activated cell sorting to purify and study control or leptin receptor-deleted (Lepr null) somatotropes. We report that Lepr-null somatotropes show significant reductions in GH protein (GH) and Gh mRNA. By contrast, enzyme immunoassays detected no changes in ACTH, LH, and FSH levels in mutants, indicating that the control of these hormones is independent of leptin signaling to somatotropes. Reduced TSH and PRL levels were also observed, but interestingly, this reduction occurred only in in Lepr-null somatotropes from mutant females and not from males. Consistent with the sex-specific reduction in Gh mRNA, TSH, and PRL, enzyme immunoassays detected a sex-specific reduction in POU1F1 protein levels in adult female Lepr-null somatotropes. Collectively, this study of purified Lepr-null somatotropes has uncovered an unexpected tropic role for leptin in the control of POU1F1 and all POU1F1-dependent hormones. This supports a broader role for somatotropes as metabolic sensors including sex-specific responses to leptin.

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress.

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.

Functional Integration of mRNA Translational Control Programs.

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.

Neural stem and progenitor cell fate transition requires regulation of Musashi1 function.

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, p21(WAF1/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.