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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 In recent years, 3D printing technology has become inexpensive and simple enough that any lab can own and use one of these printers. New method We explored the potential use of 3D printers for quickly and easily producing in vitro slice chambers for patch clamp electrophysiology. Slice chambers were produced using five available plastics: ABS, PLA, Nylon 618, Nylon 680, and T-glase. These “lab-made” chambers were also made using stereolithography through a professional printing service (Shapeways). This study measured intrinsic membrane properties of neurons in the brain stem pedunculopontine nucleus (PPN) and layer V pyramidal neurons in retrosplenial cortex. Results Nylon 680 and T-glase significantly hyperpolarized PPN neurons. ABS increased input resistance, decreased action potential amplitude, and increased firing frequency in pyramidal cortical neurons. To test long term exposure to each plastic, human neuroblastoma SHSY5Y cell cultures were exposed to each plastic for 1 week. ABS decreased cell counts while Nylon 618 and Shapeways plastics eliminated cells. Primary mouse pituitary cultures were also tested for 24-h exposure. ABS decreased cell counts while Nylon 618 and Shapeways plastics dramatically decreased cell counts. Comparison to existing methods Chambers can be quickly and inexpensively printed in the lab. ABS, PLA, Nylon 680, and T-glase plastics would suffice for many experiments instead of commercially produced slice chambers. Conclusions While these technologies are still in their infancy, they represent a powerful addition to the lab environment. With careful selection of print material, slice chambers can be quickly and inexpensively manufactured in the lab. [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]

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. [ABSTRACT FROM AUTHOR]

In response to hormonal stimulation quiescent 3T3-L1 preadipocyte cells reenter the cell cycle and undergo a mitotic expansion phase prior to terminal differentiation. The cell cycle regulatory proteins p130 and p107 undergo dramatic changes in protein levels within 24 h of differentiation. The role of these proteins in regulating adipocyte mitotic clonal expansion and/or differentiation are unclear. It has recently been demonstrated that adipocyte proliferation can be uncoupled from adipocyte differentiation through the use of the pharmacological MEK inhibitor PD98059 or the tyrosine phosphatase inhibitor, sodium vanadate. We examined the expression of p130 and p107 in stimulated 3T3-L1 cells in the presence of either PD98059, U0126 or sodium vanadate. While inhibition of MEK blocked proliferation, the cells underwent differentiation normally. In contrast, vanadate blocked differentiation without affecting proliferation. Inhibition of MEK did not affect the increase in p107 expression in stimulated cells indicating that induction of p107 is independent of MAP kinase signaling. Vanadate treatment caused a significant delay in p107 expression in the first 24 h following stimulation. Under these conditions, p130 expression was relatively unchanged. Our results indicate that a rapid increase in p107 expression correlates with a commitment to undergo adipocyte differentiation. The data further suggest that the rapid induction of p107 is not required for cellular proliferation during the mitotic clonal expansion phase. Taken together, these findings provide correlative data that implicate p107 in the terminal differentiation, but not proliferation, of quiescent preadipocytes following hormonal stimulation. [Copyright &y& Elsevier]

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]

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. [ABSTRACT FROM AUTHOR]

The developing pituitary is a rapidly changing environment that is constantly meeting the physiological demands of the growing organism. During early postnatal development, the anterior pituitary is refining patterns of anterior hormone secretion in response to numerous genetic factors. Our laboratory previously developed a somatotrope leptin receptor (LEPR) deletion mouse model that had decreased lean body mass, disrupted metabolism, decreased GH stores and was GH deficient as an adult. To understand how deletion of LEPR in somatotropes altered GH, we turned our attention to postnatal development. The current study examines GH, PRL, TSH, ACTH, LH and FSH secretion during postnatal days 4, 5, 8, 10 and 15 and compares age and sex differences. The LEPR mutants have dysregulation of GH (P < 0.03) and a reduced developmental prolactin peak in males (P < 0.04) and females (P < 0.002). There were no differences in weight between groups, and the postnatal leptin surge appeared to be normal. Percentages of immunolabeled GH cells were reduced in mutants compared with controls in all age groups by 35-61% in males and 41-44% in females. In addition, we measured pituitary expression of pituitary transcription factors, POU1F1 and PROP1. POU1F1 was reduced in mutant females at PND 10 (P < 0.009) and PND 15 (P < 0.02) but increased in males at PND 10 (P < 0.01). PROP1 was unchanged in female mutants but showed developmental increases at PND 5 (P < 0.02) and PND 15 (P < 0.01). These studies show that the dysfunction caused by LEPR deletion in somatotropes begins as early as neonatal development and involves developing GH and prolactin cells (somatolactotropes). [ABSTRACT FROM AUTHOR]

This review highlights the most important discovery in the reticular activating system in the last 10 years, the manifestation of gamma band activity in cells of the reticular activating system (RAS), especially in the pedunculopontine nucleus, which is in charge of waking and rapid eye movement (REM) sleep. The identification of different cell groups manifesting P/Q-type Ca2+ channels that control waking vs. those that manifest N-type channels that control REM sleep provides novel avenues for the differential control of waking vs. REM sleep. Recent discoveries on the development of this system can help explain the developmental decrease in REM sleep and the basic rest-activity cycle. [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]

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]

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 (IC 50 range of 1.1 ± 0.1–1.4 ± 0.2 µM) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC 50 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. [Display omitted] • Single- and double-modified salinomycin analogs were more potent towards MDA-MB-231 breast cancer cells than salinomycin • Double modified analog 17 induced DNA fragmentation and apoptotic cell death in MDA-MB-231 cell monolayer and organoids • Analog 17 reduced colony formation potential and migration of MDA-MB-231 cells to the greater extent than salinomycin • Analog 17 more effectively than salinomycin reduced CD44+/CD24- cancer stem-like cells in monolayer and organoid cultures [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Myogenic progenitors in adult muscle are necessary for the repair, maintenance and hypertrophy of post-mitotic muscle fibers. With age, fat deposition and fibrosis contribute to the decline in the integrity and functional capacity of muscles. In a previous study we reported increased accumulation of lipid in myogenic progenitors obtained from aged mice, accompanied by an up-regulation of genes involved in adipogenic differentiation. The present study was designed to extend our understanding of how aging affects the fate and gene expression profile of myogenic progenitors. Affymetrix murine U74 Genechip analysis was performed using RNA extracted from myogenic progenitors isolated from adult (8-month-old) and aged (24-month-old) DBA/2JNIA mice. The cells from the aged animals exhibited major alterations in the expression level of many genes directly or indirectly involved with the TGFβ signaling pathway. Our data indicate that with age, myogenic progenitors acquire the paradoxical phenotype of being both TGFβ activated based on overexpression of TGFβ-inducible genes, but resistant to the differentiation-inhibiting effects of exogenous TGFβ. The overexpression of TGFβ-regulated genes, such as connective tissue growth factor, may play a role in increasing fibrosis in aging muscle. [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]

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]