Activation Of mRNA Translation Research Articles

Control of Synaptic Plasticity and Memory via Suppression of Poly(A)-Binding Protein

Control of protein synthesis is critical for synaptic plasticity and memory formation. However, the molecular mechanisms linking neuronal activity to activation of mRNA translation are not fully understood. Here, we report that the translational repressor poly(A)-binding protein (PABP)-interacting protein 2A (PAIP2A), an inhibitor of PABP, is rapidly proteolyzed by calpains in stimulated neurons and following training for contextual memory. Paip2a knockout mice exhibit a lowered threshold for the induction of sustained long-term potentiation and an enhancement of long-term memory after weak training. Translation of CaMKIIα mRNA is enhanced in Paip2a⁻/⁻ slices upon tetanic stimulation and in the hippocampus of Paip2a⁻/⁻ mice following contextual fear learning. We demonstrate that activity-dependent degradation of PAIP2A relieves translational inhibition of memory-related genes through PABP reactivation and conclude that PAIP2A is a pivotal translational regulator of synaptic plasticity and memory.

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb(+/+) mice but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.

Caspase-3 cleaves hnRNP K in erythroid differentiation

Post-transcriptional control of gene expression is crucial for the control of cellular differentiation. Erythroid precursor cells loose their organelles in a timely controlled manner during terminal maturation to functional erythrocytes. Extrusion of the nucleus precedes the release of young reticulocytes into the blood stream. The degradation of mitochondria is initiated by reticulocyte 15-lipoxygenase (r15-LOX) in mature reticulocytes. At that terminal stage the release of r15-LOX mRNA from its translational silenced state induces the synthesis of r15-LOX. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key regulator of r15-LOX mRNA translation. HnRNP K that binds to the differentiation control element (DICE) in the 3′ untranslated region (UTR) inhibits r15-LOX mRNA translation initiation. During erythroid cell maturation, activation of r15-LOX mRNA translation is mediated by post-translational modifications of hnRNP K and a decrease of the hnRNP K level. To further elucidate its function in the post-transcriptional control of gene expression, we investigated hnRNP K degradation employing an inducible erythroid cell system that recapitulates both nuclear extrusion and the timely controlled degradation of mitochondria, mediated by the activation of r15-LOX synthesis. Interestingly, we detected a specific N-terminal cleavage intermediate of hnRNP K lacking DICE-binding activity that appeared during erythroid differentiation and puromycin-induced apoptosis. Employing mass spectrometry and enzymatic analyses, we identified Caspase-3 as the enzyme that cleaves hnRNP K specifically. In vitro studies revealed that cleavage by Caspase-3 at amino acids (aa) D334-G335 removes the C-terminal hnRNP K homology (KH) domain 3 that confers binding of hnRNP K to the DICE. Our data suggest that the processing of hnRNP K by Caspase-3 provides a save-lock mechanism for its timely release from the r15-LOX mRNA silencing complex and activation of r15-LOX mRNA synthesis in erythroid cell differentiation.

HIV-1 Viral Infectivity Factor (Vif) Alters Processive Single-stranded DNA Scanning of the Retroviral Restriction Factor APOBEC3G*

APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (-)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of VifIIIB- and VifHXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, VifHXB2 inhibited the jumping and VifIIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.

Heterogeneous Cationic Liposomes Modified with 3β-{N-[(N′,N′-Dimethylamino)ethyl]carbamoyl}cholesterol Can Induce Partial Conformational Changes in Messenger RNA and Regulate Translation in an Escherichia coli Cell-Free Translation System

The effect of cationic liposomes (CLs) on messenger RNA(mRNA) conformation and translation was studied, focusing on membrane heterogeneity. CLs, composed of 1,2-dioleoyl-sn-glycerol-3-phosphocholine/1,2-dioleoyl-3-timethylammonium propane (DOPC/DOTAP) and DOPC/3β-{N-[(N',N'-dimethylamino)ethyl]carbamoyl}cholesterol (DOPC/DC-Ch), inhibited mRNA translation in an Escherichia coli cell-free translation system. Analysis of the membrane fluidity and polarity indicated a heterogeneous DOPC/DC-Ch (70/30) membrane, while other CLs exhibited homogeneous disordered membranes. mRNA adsorbed onto DOPC/DC-Ch liposomes showed translational activity, while DOPC/DOTAP liposomes inhibited mRNA translation in proportion to its adsorption onto membranes. Dehydration of DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) was observed in the presence of mRNA but not in the case of zwitterionic DOPC liposomes, indicating that mRNA binds in regions between the phosphate [-PO(2)(-)-] and carbonyl [-C=O-] moieties of lipids. UV resonance Raman spectroscopy suggests that adenine, cytosine, and guanine interact with DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) but not with DOPC. Circular dichroism indicates that DOPC/DOTAP (70/30) extensively denatured the mRNA. In contrast, heterogeneous DOPC/DC-Ch (70/30) induced partial conformational changes but maintained the translational activity of mRNA.

Light-inducible activation of target mRNA translation in mammalian cells

A genetically encoded optogenetic system was constructed that activates mRNA translation in mammalian cells in response to light. Blue light induces the reconstitution of an RNA binding domain and a translation initiation domain, thereby activating target mRNA translation downstream of the binding sites.

HnRNP A1 mediates the activation of the IRES-dependent SREBP-1a mRNA translation in response to endoplasmic reticulum stress

A growing amount of evidence suggests the involvement of ER (endoplasmic reticulum) stress in lipid metabolism and in the development of some liver diseases such as steatosis. The transcription factor SREBP-1 (sterol-regulatory-element-binding protein 1) modulates the expression of several enzymes involved in lipid synthesis. Previously, we showed that ER stress increased the SREBP-1a protein level in HepG2 cells, by inducing a cap-independent translation of SREBP-1a mRNA, through an IRES (internal ribosome entry site), located in its leader region. In the present paper, we report that the hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) interacts with 5'-UTR (untranslated region) of SREBP-1a mRNA, as an ITAF (IRES trans-acting factor), regulating SREBP-1a expression in HepG2 cells and in primary rat hepatocytes. Overexpression of hnRNP A1 in HepG2 cells and in rat hepatocytes increased both the SREBP-1a IRES activity and SREBP-1a protein level. Knockdown of hnRNP A1 by small interfering RNA reduced either the SREBP-1a IRES activity or SREBP-1a protein level. hnRNP A1 mediates the increase of SREBP-1a protein level and SREBP-1a IRES activity in Hep G2 cells and in rat hepatocytes upon tunicamycin- and thapsigargin-induced ER stress. The induced ER stress triggered the cytosolic relocation of hnRNP A1 and caused the increase in hnRNP A1 bound to the SREBP-1a 5'-UTR. These data indicate that hnRNP A1 participates in the IRES-dependent translation of SREBP-1a mRNA through RNA-protein interaction. A different content of hnRNP A1 was found in the nuclei from high-fat-diet-fed mice liver compared with standard-diet-fed mice liver, suggesting an involvement of ER stress-mediated hnRNP A1 subcellular redistribution on the onset of metabolic disorders.

L-Leucine Improves the Anemia of DBA and the 5q- Syndrome Via Activation of the mTOR Pathway in a p53-Independent Manner

Abstract 1257Haploinsufficiency of ribosomal proteins (RP) has been shown to be the common basis for the anemia observed in ribosomopathies such as Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q (del(5q) MDS). DBA is a congenital bone marrow failure syndrome characterized by a profound macrocytic anemia. More than half the patients with DBA have been shown to have a heterozygous loss of an RP gene, with RPS19 being the most frequently mutated. The “5q- syndrome” is a subtype of myelodysplastic syndrome (MDS) also characterized by severe anemia that is caused by heterozygous loss of the RPS14 gene on chromosome 5q. The p53 pathway is known to play a critical role in the pathophysiology of the ribosomopathies. The leading hypothesis is that ribosomal haploinsufficiency leads to disrupted ribosome biogenesis with an accumulation of free ribosomal proteins that bind MDM2. MDM2 is an E3 Ubiquitin ligase that normally binds to and targets p53 for proteosomal degradation. The consequent accumulation of p53 leads to cell cycle arrest and apoptosis, which ultimately results in anemia. Several animal models have shown that the anemia associated with RP haploinsufficiency is almost completely alleviated in a p53 null background. However, we and others have shown that p53-independent pathway(s) also contribute to the anemia associated with RP haploinsufficiency.We have previously modeled DBA and del (5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14 respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. We have further demonstrated that treatment of Rps19 and Rps14 deficient embryos with the amino acid L-Leucine, a known activator of mRNA translation, results in a marked improvement in anemia. This observation was confirmed in primary human CD34+ cells, following shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that L-leucine treatment activates the mTOR pathway in zebrafish embryos deficient in Rps19 or Rps14. In order to determine if the effect of L-Leucine on RP deficient erythroid cells is p53 dependent, we injected rps19 and rps14 morpholinos into zebrafish embryos and treated them with L-Leucine. Total RNA was collected 48hpf and evaluated for expression of p53 by qPCR analysis. As expected, the expression of p53 and its downstream targets (p21 and PUMA) were upregulated in Rps14 and Rps19 deficient embryos. P53 expression levels remained elevated even after L-Leucine treatment. Levels of p21, a direct transcriptional target of p53, remained unchanged in L-Leucine treated RP deficient zebrafish embryos; however, expression of PUMA increased following L-Leucine treatment. The expression of the PUMA gene has previously been shown to have a p53-independent regulatory component. Preliminary studies in the A549 cell line, which harbors wild type p53, also showed increased levels of p53 expression upon shRNA mediated downregulation of both RPS19 and RPS14, which remained unaltered following L-Leucine treatment. These observations are currently being confirmed in primary human CD34+ cells, following shRNA knockdown of RPS19 and RPS14.Our preliminary studies show that the effect of L-Leucine in improving the anemia in models of DBA and del(5q) MDS occurs independently of p53. This supports our hypothesis that the erythroid phenotype in these disorders has a p53-independent component. Our finding that L-Leucine treated RP deficient cells are likely to express elevated rather than diminished levels of p53 in spite of improved anemia also has important implications for the clinical management of patients, since p53 inactivation is associated with tumor growth. A trial using L-Leucine for patients with DBA will be opening soon in the United States. Disclosures:Ebert:Celgene: Consultancy; Genoptix: Consultancy.

MicroRNA-mediated mRNA Translation Activation in Quiescent Cells and Oocytes Involves Recruitment of a Nuclear microRNP

MicroRNAs can promote translation of specific mRNAs in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes. We report that microRNA-mediated upregulation of target mRNAs in oocytes is dependent on nuclear entry of the microRNA; cytoplasmically-injected microRNA repress target mRNAs. Components of the activation microRNP, AGO, FXR1 (FXR1-iso-a) and miR16 are present in the nucleus and cytoplasm. Importantly, microRNA target mRNAs for upregulation, Myt1, TNFα and a reporter bearing the TNFα AU-rich, microRNA target sequence, are associated with AGO in immature oocyte nuclei and AGO2 in G0 human nuclei, respectively. mRNAs that are repressed or lack target sites are not associated with nuclear AGO. Crosslinking-coupled immunopurification revealed greater association of AGO2 with FXR1 in the nucleus compared to cytoplasm. Consistently, overexpression of FXR1-iso-a rescues activation of cytoplasmically-injected RNAs and in low density, proliferating cells. These data indicate the importance of a compartmentalized AGO2-FXR1-iso-a complex for selective recruitment for microRNA-mediated upregulation.

L-leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway

AbstractHaploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34+ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.

Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation

HIF-2alpha plays a critical role in renal tumorigenesis. HIF-2alpha is stabilized in Von Hippel-Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of Rapamycin (mTOR) functions in two distinct complexes, Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2alpha protein in the absence of VHL, however the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation, is not clear. Complex assembly of the mRNA cap protein, eIF4E, with activators (eIF4G) and inhibitors (4E-BP1) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22phox-based Nox oxidases, are enhanced in VHL-deficient cells and play a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2alpha through eIF4E-dependent mRNA translation and examined the effects of p22phox-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive and further defined a novel role for p22phox-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22phox reduces HIF-2alpha-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

New perspectives in the control of body protein metabolism

Recent advances in elucidating the mechanisms that control body protein synthesis and degradation both expand and complicate our understanding of how these processes are regulated. This review presents an introduction to the multiple regulatory systems involved, emphasizing the number of potential controls. These include gene transcription, gene activation or suppression, activation or suppression of mRNA translation and activation or suppression of signaling pathways. The complexity of these interacting controls presents a challenge to our understanding of the overall coordinated regulation of protein synthesis and degradation and its response to any particular stimulus. Specific examples are used to illustrate regulatory mechanisms, including the ways in which protein metabolism is regulated by the amino acid leucine. In addition to regulation associated with gene expression and post-translational control, the expanding field of epigenetics adds another layer of complexity, including trans-generational responses to nutrient intake, highlighting the potential for long-term impact of nutritional experience on the metabolism of subsequent generations.

Quick regulation of mRNA functions by a few seconds of photoirradiation

3-Cyanovinylcarbazole nucleoside, which effectively photocrosslinks to the pyrimidine base in complementary RNA strands, was incorporated into antisense oligonucleotides, and we evaluated the photoreactivity and the sequence selectivity to mutated K-ras oligoRNAs, as well as the regulation of the function of K-ras mRNA. We demonstrated that the reverse transcription and the translation activity of K-ras mRNA were quickly suppressed by a few seconds of photoirradiation with the addition of the photoresponsive antisense ODN.

Abstract P111: Rheb-mTOR Signaling Pathway Regulates Cardiomyocyte Mass During Post-Neonatal Period

Rheb (Ras homologue enriched in brain) is a major activator of mTOR. Rheb-mTOR pathway is a critical mechanism for maintenance of homeostasis, cell growth and stress response by regulating both protein synthesis and degradation. In this study, we attempted to clarify the role of Rheb-mTOR pathway in the heart using cardiac-specific Rheb-deficient mice (Rheb −/− ). We generated floxed Rheb mice and crossed them with transgenic mice expressing Cre recombinase in cardiac-specific mannner to generate Rheb −/− . Rheb −/− were born in Mendelian ratio, but they started to die 8 days after birth and all of them had died until 10 days after birth. Echocardiographic analysis revealed that chamber dimension and contractile function of Rheb −/− were indistinguishable from those of control mice (Rheb +/+ ) 5 days after birth. However, Rheb −/− exhibited cardiac dilatation and reduced contractility 8 days after birth (LV end diastolic dimension, Rheb −/− : 2.5±0.2 mm vs. Rheb +/+ : 2.1±0.2 mm, p<0.01, fractional shortening, Rheb −/− : 19.7 ± 9.7 % vs. Rheb +/+ : 48.6 ± 8.8 %, p<0.01). These suggest that Rheb −/− died of cardiac dysfunction and heart failure. Heart weight and cross-sectional area of cardiomyocytes were significantly lower in Rheb −/− 8 days after birth. Electron microscopic analysis revealed that the area of sarcomere was significantly lower in Rheb −/− cardiomyocytes. Expressions of sarcomeric proteins, such as myosin heavy chain, actin or desmin, were decreased in Rheb −/− , while the mRNA expression of desmin was significantly increased in Rheb −/− . Thus impairment of cardiomyocyte growth observed in Rheb −/− could be due to either increased degradation or decreased translation. Although autophagic activity was enhanced in Rheb −/− heart, ablation of Atg5, an essential molecule for autophagy, could not prevent premature death of Rheb −/− . On the other hand, polysome analysis revealed that the mRNA translation activity had decreased in Rheb −/− heart compared with Rheb +/+ . Thus, we concluded that Rheb-mTOR pathway in the heart is essential to regulate mRNA translation activity and protein synthesis, thereby to cardiomyocyte growth in neonatal period.

Key contribution of CPEB4-mediated translational control to cancer progression.

Malignant transformation, invasion and angiogenesis rely on the coordinated reprogramming of gene expression in the cells from which the tumor originated. Although deregulated gene expression has been extensively studied at genomic and epigenetic scales, the contribution of the regulation of mRNA-specific translation to this reprogramming is not well understood. Here we show that cytoplasmic polyadenylation element binding protein 4 (CPEB4), an RNA binding protein that mediates meiotic mRNA cytoplasmic polyadenylation and translation, is overexpressed in pancreatic ductal adenocarcinomas and glioblastomas, where it supports tumor growth, vascularization and invasion. We also show that, in pancreatic tumors, the pro-oncogenic functions of CPEB4 originate in the translational activation of mRNAs that are silenced in normal tissue, including the mRNA of tissue plasminogen activator, a key contributor to pancreatic ductal adenocarcinoma malignancy. Taken together, our results document a key role for post-transcriptional gene regulation in tumor development and describe a detailed mechanism for gene expression reprogramming underlying malignant tumor progression.

P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,

Abstract 3432Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation. Disclosures:No relevant conflicts of interest to declare.

Embryonic poly(A) binding protein (ePAB)-deficient male mice have elevated MSY2 expression in testis

Translational activation of mRNAs stored in the cytoplasm is a key process in the regulation of gene expression during spermatogenesis. Embryonic poly(A) binding protein (ePAB) is the predominant poly(A) binding protein during oocyte and early embryo development until ZGA; it stabilizes mRNAs and promotes their translation. We hypothesized that ePAB plays a key role in spermatogenesis and tested our hypothesis by generating and characterizing mice homozygous-deficient for ePAB.

Reversible Inhibition of PSD-95 mRNA Translation by miR-125a, FMRP Phosphorylation, and mGluR Signaling

The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 are reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.

Mechanism of Mss116 ATPase Reveals Functional Diversity of DEAD-Box Proteins

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (Pi) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-Pi binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116–ADP-Pi state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with Pi release dissociates Mss116–RNA complexes, regenerating free Mss116.

Biochemical Characterization of Pumilio1 and Pumilio2 in Xenopus Oocytes

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.