Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events that are driving these transitions. Here we used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA-binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation. Using this strategy, we identified highly specific patterns of mRNA recruitment to the polysomes that are synchronized with the cell cycle. A large number of the mRNAs recovered with translating ribosomes contain motifs for the RNA-binding proteins DAZL (deleted in azoospermia-like) and CPEB (cytoplasmic polyadenylation element-binding protein). Although a Dazl role in early germ cell development is well established, no function has been described during oocyte-to-embryo transition. We demonstrate that CPEB1 regulates Dazl post-transcriptionally, and that DAZL is essential for meiotic maturation and embryonic cleavage. In the absence of DAZL synthesis, the meiotic spindle fails to form due to disorganization of meiotic microtubules. Therefore, Cpeb1 and Dazl function in a progressive, self-reinforcing pathway to promote oocyte maturation and early embryonic development.

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The mechanism by which the 8q24 MYC enhancer region, including cancer-associated variant rs6983267, increases cancer risk is unknown due to the lack of protein-coding genes at 8q24.21. Here we report the identification of long noncoding RNAs named cancer-associated region long noncoding RNAs (CARLos) in the 8q24 region. The expression of one of the long noncoding RNAs, CARLo-5, is significantly correlated with the rs6983267 allele associated with increased cancer susceptibility. We also found the MYC enhancer region physically interacts with the active regulatory region of the CARLo-5 promoter, suggesting long-range interaction of MYC enhancer with the CARLo-5 promoter regulates CARLo-5 expression. Finally, we demonstrate that CARLo-5 has a function in cell-cycle regulation and tumor development. Overall, our data provide a key of the mystery of the 8q24 gene desert.

Keeping Wee1B in the nucleus is important to maintain meiotic arrest, but its timely export is also required for meiosis to resume.

The fully grown mammalian oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during early development. However, we find that an abundant RNA population is retained in the oocyte nucleus and contains specific mRNAs important for meiotic progression. Here we show that during the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region that was previously surrounded the nucleus. These distinct translational hotspots are separated by endoplasmic reticulum and Lamin, and disappear following polar body extrusion. Chromosomal translational hotspots are controlled by the activity of the mTOR–eIF4F pathway. Here we reveal a mechanism that—following the resumption of meiosis—controls the temporal and spatial translation of a specific set of transcripts required for normal spindle assembly, chromosome alignment and segregation.

Waves of cyclin synthesis and degradation regulate the activity of Cdc2 protein kinase during the cell cycle. Cdc2 inactivation by Wee1B-mediated phosphorylation is necessary for arrest of the oocyte at G2-prophase, but it is unclear whether this regulation functions later during the metaphase to anaphase transition. We show that reactivation of a Wee1B pathway triggers the decrease in Cdc2 activity during egg activation. When Wee1B is downregulated, oocytes fail to form a pronucleus in response to Ca2+ signals. Calcium-calmodulin-dependent kinase II (CaMKII) activates Wee1B, and CaMKII-driven exit from metaphase II (MII) is inhibited by Wee1B downregulation, demonstrating that exit from metaphase requires not only a proteolytic degradation of cyclin B, but also the inhibitory phosphorylation of Cdc2 by Wee1B.

synGAP is a neuron-specific Ras GTPase-activating protein found in high concentration in the postsynaptic density fraction from mammalian forebrain. Proteins in the postsynaptic density, including synGAP, are part of a signaling complex attached to the cytoplasmic tail of the N-methyl-D-aspartate-type glutamate receptor. syn-GAP can be phosphorylated by a second prominent component of the complex, Ca 2؉ /calmodulin-dependent protein kinase II. Here we show that phosphorylation of synGAP by Ca 2؉ /calmodulin-dependent protein kinase II increases its Ras GTPase-activating activity by 70 -95%. We identify four major sites of phosphorylation, serines 1123, 1058, 750/751/756, and 764/765. These sites together with other minor phosphorylation sites in the carboxyl tail of synGAP control stimulation of GTPaseactivating activity. When three of these sites and four other serines in the carboxyl tail are mutated, stimulation of GAP activity after phosphorylation is reduced to 21 ؎ 5% compared with 70 -95% for the wild type protein.We used phosphosite-specific antibodies to show that, as predicted, phosphorylation of serines 765 and 1123 is increased in cultured cortical neurons after exposure of the neurons to the agonist N-methyl-D-aspartate.Storage of information in the brain is mediated in part by changes in the strength of synaptic connections between neurons initiated by specific patterns of electrical activity (1). These changes involve complex regulatory pathways that are controlled by the pattern of influx of Ca 2ϩ ion through Nmethyl-D-aspartate (NMDA) 1 -type glutamate receptors (NMDA receptors) at postsynaptic sites. Much present research concerns the nature of the relevant biochemical pathways and the mechanisms of Ca 2ϩ control. One set of regulatory proteins associates tightly with the cytosolic portion of the NMDA receptor (2, 3). These include Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) (4, 5), which is activated by Ca 2ϩ flux through the receptor, and several proteins that are held near the receptor by the scaffold protein PSD-95 (6, 7). A complex containing many of these proteins can be isolated from brain homogenates and is called the postsynaptic density (PSD) fraction (8).synGAP was identified as a prominent 140-kDa protein in the PSD fraction (9, 10) and as a protein that interacts with PSD-95 in a yeast two-hybrid screen (11). Its message is detected only in brain (10, 11). It is expressed only in neurons, including most excitatory neurons and a subset of inhibitory neurons (12), where it is highly localized to the postsynaptic density (10). It contains a PH domain, a C2 domain, and a Ras GAP domain that are 23%, 33%, and 47% similar, respectively, to those of the prototype Ras GAP protein p120 Ras GAP (13). In brain homogenates, synGAP is tightly bound to the particulate fraction and full-length synGAP has not yet been purified in soluble form; however, the GAP domain, expressed as a GST fusion protein in Escherichia coli, has been shown to stimulate hydrolysis of bound GTP by Ras (11). ...

Abnormal cell-cycle control can lead to aberrant cell proliferation and cancer. The oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) is an inhibitor of protein phosphatase 2A (PP2A) that stabilizes c-Myc. However, the precise role of CIP2A in cell division is not understood. Herein, we show that CIP2A is required for mitotic progression by regulating the polo-like kinase (Plk1). With mitotic entry, CIP2A translocated from the cytoplasm to the nucleus, where it was enriched at spindle poles. CIP2A depletion delayed mitotic progression, resulting in mitotic abnormalities independent of PP2A activity. Unexpectedly, CIP2A interacted directly with the polo-box domain of Plk1 during mitosis. This interaction was required to maintain Plk1 stability by blocking APC/C-Cdh1-dependent proteolysis, thereby enhancing the kinase activity of Plk1 during mitosis. We observed strong correlation and in vivo interactions between these two proteins in multiple human cancer specimens. Overall, our results established a novel function for CIP2A in facilitating the stability and activity of the pivotal mitotic kinase Plk1 in cell-cycle progression and tumor development. Cancer Res; 73(22); 6667-78. Ó2013 AACR.