Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.

Temporal and spatial control of mRNA translation has emerged as a major mechanism for promoting diverse biological processes. However, the molecular nature of temporal and spatial control of translation remains unclear. In oocytes, many mRNAs are deposited as a translationally repressed form and are translated at appropriate times to promote the progression of meiosis and development. Here, we show that changes in subcellular structures and states of the RNA-binding protein pumilio 1 (Pum1) regulate the translation of target mRNAs and progression of oocyte maturation. Pum1 was shown to bind to Mad2 (also known as Mad2l1) and cyclin B1 mRNAs, assemble highly clustered aggregates, and surround Mad2 and cyclin B1 RNA granules in mouse oocytes. These Pum1 aggregates were dissolved prior to the translational activation of target mRNAs, possibly through phosphorylation. Stabilization of Pum1 aggregates prevented the translational activation of target mRNAs and progression of oocyte maturation. Together, our results provide an aggregation-dissolution model for the temporal and spatial control of translation.

Vitrification-induced activation of lysosomal cathepsin B perturbs spindle assembly checkpoint function in mouse oocytes.

As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus-oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.

Posttranscriptional regulation of maternal Pou5f1/Oct4 during mouse oogenesis and early embryogenesis.

Protein syntheses at appropriate timings are important for promoting diverse biological processes and are controlled at the levels of transcription and translation. Pou5f1/Oct4 is a transcription factor that is essential for vertebrate embryonic development. However, the precise timings when the mRNA and protein of Pou5f1/Oct4 are expressed during oogenesis and early stages of embryogenesis remain unclear. We analyzed the expression patterns of mRNA and protein of Pou5f1/Oct4 in mouse oocytes and embryos by using a highly sensitive in situ hybridization method and a monoclonal antibody specific to Pou5f1/Oct4, respectively. Pou5f1/Oct4 mRNA was detected in growing oocytes from the primary follicle stage to the fully grown GV stage during oogenesis. In contrast, Pou5f1/Oct4 protein was undetectable during oogenesis, oocyte maturation and the first cleavage stage but subsequently became detectable in the nuclei of early 2-cell-stage embryos. Pou5f1/Oct4 protein at this stage was synthesized from maternal mRNAs stored in oocytes. The amount of Pou5f1/Oct4 mRNA in the polysomal fraction was small in GV-stage oocytes but was significantly increased in fertilized eggs. Taken together, our results indicate that the synthesis of Pou5f1/Oct4 protein during oogenesis and early stages of embryogenesis is controlled at the level of translation and suggest that precise control of the amount of this protein by translational regulation is important for oocyte development and early embryonic development.

Dynamic whole-body 18F-FDG PET for differentiating abnormal lesions from physiological uptake.

PURPOSE: Serial assessment of visual change in 18F-FDG uptake on whole-body 18F-FDG PET imaging was performed to differentiate pathological uptake from physiological uptake in the urinary and gastrointestinal tracts. METHODS: In 88 suspected cancer patients, serial 3-min dynamic whole-body PET imaging was performed four times, from 60 min after 18F-FDG administration. In dynamic image evaluation, high 18F-FDG uptake was evaluated by two nuclear medicine physicians and classified as "changed" or "unchanged" based on change in uptake shape over time. Detectability of pathological uptake based on these criteria was assessed and compared with conventional image evaluation. RESULTS: Dynamic whole-body PET imaging provided images of adequate quality for visual assessment. Dynamic image evaluation was "changed" in 118/154 regions of high physiological 18F-FDG uptake (77%): in 9/19 areas in the stomach (47%), in 32/39 in the small intestine (82%), in 17/33 in the colon (52%), and in 60/63 in the urinary tract (95%). In the 86 benign or malignant lesions, 84 lesions (98%) were "unchanged." A high 18F-FDG uptake area that shows no change over time using these criteria is highly likely to represent pathological uptake, with sensitivity of 97%, specificity of 76%, PPV of 70%, NPV of 98%, and accuracy of 84%. CONCLUSION: Dynamic whole-body 18F-FDG PET imaging enabled differentiation of pathological uptake from physiological uptake in the urinary and gastrointestinal tracts, based on visual change of uptake shape.

A novel testis-specific long noncoding RNA, Tesra, activates the Prss42/Tessp-2 gene during mouse spermatogenesis†.

The progression of spermatogenesis is precisely controlled by meiotic stage-specific genes, but the molecular mechanism for activation of such genes is still elusive. Here we found a novel testis-specific long noncoding RNA (lncRNA), Tesra, that was specifically expressed in the mouse testis at the Prss/Tessp gene cluster on chromosome 9. Tesra was transcribed downstream of Prss44/Tessp-4, starting within the gene, as a 4435-nucleotide transcript and developmentally activated at a stage similar to that for Prss/Tessp genes. By in situ hybridization, Tesra was found to be localized in and around germ cells and Leydig cells, being consistent with biochemical data showing its existence in cytoplasmic, nuclear, and extracellular fractions. Based on the finding of more signals in nuclei of pachytene spermatocytes, we explored the possibility that Tesra plays a role in transcriptional activation of Prss/Tessp genes. By a ChIRP assay, the Tesra transcript was found to bind to the Prss42/Tessp-2 promoter region in testicular germ cells, and transient overexpression of Tesra significantly activated endogenous Prss42/Tessp-2 expression and increased Prss42/Tessp-2 promoter activity in a reporter construct. These findings suggest that Tesra activates the Prss42/Tessp-2 gene by binding to the promoter. Finally, we investigated whether Tesra co-functioned with enhancers adjacent to another lncRNA, lncRNA-HSVIII. In the Tet-on system, Tesra transcription significantly increased activity of one enhancer, but Tesra and the enhancer were not interdependent. Collectively, our results proposed a potential function of an lncRNA, Tesra, in transcriptional activation and suggest a novel relationship between an lncRNA and an enhancer.

Function of leukaemia inhibitory factor in spermatogenesis of a teleost fish, the medaka Oryzias latipes.

In response to gonadotropins and androgens, testicular cells produce various molecules that control proper proliferation and differentiation of spermatogenic cells through their paracrine and autocrine actions. However, molecules functioning downstream of the hormonal stimulation are poorly understood. Leukaemia inhibitory factor (Lif) is known to maintain the pluripotency of stem cells including embryonic stem cells and primordial germ cells at least in vitro, but its actual roles in vivo remain to be elucidated. To clarify the function of Lif in teleost (medaka) testes, we examined the effects of Lif on spermatogenesis in a newly established cell culture system using a cell line (named Mtp1) derived from medaka testicular somatic cells as feeder cells. We found that addition of baculovirus-produced recombinant medaka Lif to the culture medium or co-culture with Lif-overexpressing Mtp1 cells increased the number of spermatogonia. In situ hybridization and immunohistochemical analyses of the medaka testes showed that mRNAs and proteins of Lif are expressed in spermatogonia and the surrounding Sertoli cells, with higher expression levels in type A (undifferentiated) spermatogonia than in type B (differentiated) spermatogonia. Our findings suggest that Lif regulates spermatogonial cell proliferation in the medaka.

High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes.

BACKGROUND: Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method. RESULTS: The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs. CONCLUSIONS: This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.

Pumilio1 phosphorylation precedes translational activation of its target mRNA in zebrafish oocytes.

SummaryTranslational regulation of mRNAs is crucial for promoting various cellular and developmental processes. Pumilio1 (Pum1) has been shown to play key roles in translational regulation of target mRNAs in many systems of diverse organisms. In zebrafish immature oocytes, Pum1 was shown to bind to cyclin B1 mRNA and promote the formation of cyclin B1 RNA granules. This Pum1-mediated RNA granule formation seemed critical to determine the timing of translational activation of cyclin B1 mRNA during oocyte maturation, leading to activation of maturation/M-phase-promoting factor (MPF) at the appropriate timing. Despite its fundamental importance, the mechanisms of translational regulation by Pum1 remain elusive. In this study, we examined the phosphorylation of Pum1 as a first step to understand the mechanisms of Pum1-mediated translation. SDS-PAGE analyses and phosphatase treatments showed that Pum1 was phosphorylated at multiple sites during oocyte maturation. This phosphorylation began in an early period after induction of oocyte maturation, which preceded the polyadenylation of cyclin B1 mRNA. Interestingly, depolymerization of actin filaments in immature oocytes caused phosphorylation of Pum1, disassembly of cyclin B1 RNA granules, and polyadenylation of cyclin B1 mRNA but not translational activation of the mRNA. Overexpression of the Pum1 N-terminus prevented the phosphorylation of Pum1, disassembly of cyclin B1 RNA granules, and translational activation of the mRNA even after induction of oocyte maturation. These results suggest that Pum1 phosphorylation in the early period of oocyte maturation is one of the key processes for promoting the disassembly of cyclin B1 RNA granules and translational activation of target mRNA.

A genomic region transcribed into a long noncoding RNA interacts with the Prss42/Tessp-2 promoter in spermatocytes during mouse spermatogenesis, and its flanking sequences can function as enhancers.

Spermatogenesis is regulated by many meiotic stage-specific genes, but how they coordinate the many individual processes is not fully understood. The Prss/Tessp gene cluster is located on mouse chromosome 9F2-F3, and the three genes at this site (Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4) are specifically activated during meiosis in pachytene spermatocytes. We searched for DNase I hypersensitive sites (HSs) and long noncoding RNAs (lncRNAs) at the Prss/Tessp locus to elucidate how they are activated. We found eight DNase I HSs, three of which were testis germ cell-specific at or close to the Prss42/Tessp-2 promoter, and a testis-specific lncRNA, lncRNA-HSVIII, that was transcribed from a region adjacent to the Prss42/Tessp-2 gene. lncRNA-HSVIII transcripts localized to nuclei of most pachytene spermatocytes and the cytosol of stage-X pachytene spermatocytes and spermatids. Chromosome conformation capture revealed that the lncRNA-HSVIII locus specifically interacted with the Prss42/Tessp-2 promoter in primary and secondary spermatocytes. A 5.8-kb genome sequence, encompassing the entire lncRNA-HSVIII sequence and its flanking regions, significantly increased Prss42/Tessp-2 promoter activity using a reporter-gene assay, yet this construct did not change lncRNA-HSVIII expression, indicating that the elevated promoter activity was likely through enhancer activity. Indeed, both upstream and downstream regions of the lncRNA-HSVIII sequence significantly increased Prss42/Tessp-2 promoter activity. Our data therefore identified the direct interaction of a genomic region in the lncRNA-HSVIII locus with the Prss42/Tessp-2 promoter in spermatocytes, and suggested that sequences adjacent to the lncRNA function as enhancers for the Prss42/Tessp-2 gene. Mol. Reprod. Dev. 83: 541-557, 2016. © 2016 Wiley Periodicals, Inc.

A cis-acting element in the coding region of cyclin B1 mRNA couples subcellular localization to translational timing.

Subcellular localization of messenger RNAs (mRNAs) to correct sites and translational activation at appropriate timings are crucial for normal progression of various biological events. However, a molecular link between the spatial regulation and temporal regulation remains unresolved. In immature zebrafish oocytes, translationally repressed cyclin B1 mRNA is localized to the animal polar cytoplasm and its temporally regulated translational activation in response to a maturation-inducing hormone is essential to promote oocyte maturation. We previously reported that the coding region of cyclin B1 mRNA is required for the spatio-temporal regulation. Here, we report that a sequence, CAGGAGACC, that is conserved in the coding region of vertebrate cyclin B1 mRNA is involved in the regulation. Like endogenous cyclin B1 mRNA, reporter mRNAs harboring the sequence CAGGAGACC were localized to the animal polar cytoplasm of oocytes, while those carrying mutations in the sequence (with no change in the coding amino acids) were dispersed in the animal hemisphere of oocytes. Furthermore, translational activation of the mutant mRNAs was initiated at a timing earlier than that of endogenous and wild-type reporter mRNAs during oocyte maturation. Interaction of CAGGAGACC with proteins in vitro suggests that this sequence functions in collaboration with a trans-acting protein factor(s) in oocytes. These findings reveal that the sequence in the coding region of cyclin B1 mRNA plays an important role as a cis-acting element in both subcellular localization and translational timing of mRNA, providing a direct molecular link between the spatial and temporal regulation of mRNA translation.

Possible involvement of insulin-like growth factor 2 mRNA-binding protein 3 in zebrafish oocyte maturation as a novel cyclin B1 mRNA-binding protein that represses the translation in immature oocytes.

In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3' untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3' UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 mRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumilio1 and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation.

Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish.

Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.

Visualizing the translational activation of a particular mRNA in zebrafish embryos using in situ hybridization and proximity ligation assay.

Fertilized eggs initiate translation of stored mRNAs in spatially and temporally controlled manners. Here, we present a protocol for visualizing spatial and temporal translation in zebrafish embryos by fluorescence in situ hybridization and proximity ligation assay. We describe steps for labeling newly synthesized proteins and mRNA, visualizing mRNA translation and mRNA, sample mounting, and observation. Coupling detection of mRNA molecules with their translation sites is useful for understanding the molecular and cellular mechanisms that drive embryo development. For complete details on the use and execution of this protocol, please refer to Sato et al.1 and Takada et al.2.

Four-dimensional quantitative analysis using FDG-PET in clinical oncology.

Positron emission tomography (PET) with F-18 fluorodeoxyglucose (FDG) has been commonly used in many oncological areas. High-resolution PET permits a three-dimensional analysis of FDG distributions on various lesions in vivo, which can be applied for tissue characterization, risk analysis, and treatment monitoring after chemoradiotherapy and immunotherapy. Metabolic changes can be assessed using the tumor absolute FDG uptake as standardized uptake value (SUV) and metabolic tumor volume (MTV). In addition, tumor heterogeneity assessment can potentially estimate tumor aggressiveness and resistance to chemoradiotherapy. Attempts have been made to quantify intratumoral heterogeneity using radiomics. Recent reports have indicated the clinical feasibility of a dynamic FDG PET-computed tomography (CT) in pilot cohort studies of oncological cases. Dynamic imaging permits the assessment of temporal changes in FDG uptake after administration, which is particularly useful for differentiating pathological from physiological uptakes with high diagnostic accuracy. In addition, several new parameters have been introduced for the in vivo quantitative analysis of FDG metabolic processes. Thus, a four-dimensional FDG PET-CT is available for precise tissue characterization of various lesions. This review introduces various new techniques for the quantitative analysis of FDG distribution and glucose metabolism using a four-dimensional FDG analysis with PET-CT. This elegant study reveals the important role of tissue characterization and treatment strategies in oncology.

Utility of hyperdense whirl sign for the diagnosis of gallbladder torsion.

The purpose of this report was to evaluate the usefulness of hyperdense whirl sign on unenhanced computed tomography (CT) for diagnosing gallbladder torsion. The CT scans of seven patients with gallbladder torsion were independently reviewed by two board-certified radiologists for locating the high-density core with twisting between the gallbladder neck and liver bed, termed hyperdense whirl sign. The sign was observed in six cases. The detection of a hyperdense whirl sign on unenhanced CT appears useful for diagnosing gallbladder torsion.

Evaluation of Data-Driven Respiration Gating in Continuous Bed Motion in Lung Lesions.

Respiration gating is used in PET to prevent image quality degradation due to respiratory effects. In this study, we evaluated a type of data-driven respiration gating for continuous bed motion, OncoFreeze AI, which was implemented to improve image quality and the accuracy of semiquantitative uptake values affected by respiratory motion. Methods: 18F-FDG PET/CT was performed on 32 patients with lung lesions. Two types of respiration-gated images (OncoFreeze AI with data-driven respiration gating, device-based amplitude-based OncoFreeze with elastic motion compensation) and ungated images (static) were reconstructed. For each image, we calculated SUV and metabolic tumor volume (MTV). The improvement rate (IR) from respiration gating and the contrast-to-noise ratio (CNR), which indicates the improvement in image noise, were also calculated for these indices. IR was also calculated for the upper and lower lobes of the lung. As OncoFreeze AI assumes the presence of respiratory motion, we examined quantitative accuracy in regions where respiratory motion was not present using a 68Ge cylinder phantom with known quantitative accuracy. Results: OncoFreeze and OncoFreeze AI showed similar values, with a significant increase in SUV and decrease in MTV compared with static reconstruction. OncoFreeze and OncoFreeze AI also showed similar values for IR and CNR. OncoFreeze AI increased SUVmax by an average of 18% and decreased MTV by an average of 25% compared with static reconstruction. From the IR results, both OncoFreeze and OncoFreeze AI showed a greater IR from static reconstruction in the lower lobe than in the upper lobe. OncoFreeze and OncoFreeze AI increased CNR by 17.9% and 18.0%, respectively, compared with static reconstruction. The quantitative accuracy of the 68Ge phantom, assuming a region of no respiratory motion, was almost equal for the static reconstruction and OncoFreeze AI. Conclusion: OncoFreeze AI improved the influence of respiratory motion in the assessment of lung lesion uptake to a level comparable to that of the previously launched OncoFreeze. OncoFreeze AI provides more accurate imaging with significantly larger SUVs and smaller MTVs than static reconstruction.

Mature mRNA processing that deletes 3' end sequences directs translational activation and embryonic development.

Eggs accumulate thousands of translationally repressed mRNAs that are translated into proteins after fertilization to direct diverse developmental processes. However, molecular mechanisms underlying the translation of stored mRNAs after fertilization remain unclear. Here, we report a previously unknown RNA processing of 3' end sequences of mature mRNAs that activates the translation of stored mRNAs. Specifically, 9 to 72 nucleotides at the 3' ends of zebrafish pou5f3 and mouse Pou5f1 mRNAs were deleted in the early stages of development. Reporter assays illustrated the effective translation of the truncated forms of mRNAs. Moreover, promotion and inhibition of the shortening of 3' ends accelerated and attenuated Pou5f3 accumulation, respectively, resulting in defective development. Identification of proteins binding to unprocessed and/or processed mRNAs revealed that mRNA shortening acts as molecular switches. Comprehensive analysis revealed that >250 mRNAs underwent this processing. Therefore, our results provide a molecular principle that triggers the translational activation and directs development.

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.

Identification of embryonic RNA granules that act as sites of mRNA translation after changing their physical properties.

Fertilized eggs begin to translate mRNAs at appropriate times and placements to control development, but how the translation is regulated remains unclear. Here, we found that pou5f3 mRNA encoding a transcriptional factor essential for development formed granules in a dormant state in zebrafish oocytes. Although the number of pou5f3 granules remained constant, Pou5f3 protein accumulated after fertilization. Intriguingly, signals of newly synthesized peptides and a ribosomal protein became colocalized with pou5f3 granules after fertilization and, moreover, nascent Pou5f3 was shown to be synthesized in the granules. This functional change was accompanied by changes in the state and internal structure of granules. Dissolution of the granules reduced the rate of protein synthesis. Similarly, nanog and sox19b mRNAs in zebrafish and Pou5f1/Oct4 mRNA in mouse assembled into granules. Our results reveal that subcellular compartments, termed embryonic RNA granules, function as activation sites of translation after changing physical properties for directing vertebrate development.

Possible involvement of Nemo-like kinase 1 in Xenopus oocyte maturation as a kinase responsible for Pumilio1, Pumilio2, and CPEB phosphorylation.

Members of the mitogen-activated protein kinase (MAPK) family play important roles in Xenopus oocyte maturation. Nemo-like kinase (NLK), an atypical MAPK, is known to function in multiple developmental processes in vertebrates and invertebrates, but its involvement in gametogenesis and gamete maturation is unknown. In this study, we biochemically examined NLK1 during Xenopus oocyte maturation. NLK1 is expressed in immature oocytes, and its protein level remains constant during maturation. NLK1 is inactive in immature oocytes but is activated during maturation, depending on Mos protein synthesis but not on p42 MAPK activation. Overexpression of NLK1 by injection of 5 ng of mRNA accelerates progesterone-induced oocyte maturation by enhancing Cyclin B1 protein synthesis through the translational activation of its mRNA, in accordance with precocious phosphorylation of Pumilio1 (Pum1), Pumilio2 (Pum2), and cytoplasmic polyadenylation element-binding protein (CPEB), key regulators of the translational control of mRNAs stored in oocytes. A higher level of NLK1 expression by injection of 50 ng of mRNA induces Pum1/Pum2/CPEB phosphorylation, CPEB degradation, Cyclin B1 protein synthesis, and oocyte maturation in the absence of progesterone. NLK1 phosphorylates Pum1, Pum2, and CPEB in vitro. These findings provide the first evidence for the involvement of NLK1 in Xenopus oocyte maturation. We suggest that NLK1 acts as a kinase downstream of Mos and catalyzes phosphorylation of Pum1, Pum2, and CPEB to regulate the translation of mRNAs, including Cyclin B1 mRNA, stored in oocytes.