Translation In Oocytes Research Articles

The Scd6/Lsm14 protein xRAPB has properties different from RAP55 in selecting mRNA for early translation or intracellular distribution in Xenopus oocytes

Oocytes accumulate mRNAs in the form of maternal ribonucleoprotein (RNP) particles, the protein components of which determine the location and stability of individual mRNAs prior to translation. Scd6/Lsm14 proteins, typified by RAP55, function in a wide range of eukaryotes in repressing translation and relocating mRNPs to processing bodies and stress granules. In Xenopus laevis, the RAP55 orthologue xRAPA fulfils these functions. Here we describe the properties of a variant of xRAPA, xRAPB, which is a member of the Lsm14B group. xRAPB differs from xRAPA in various respects: it is expressed at high concentration earlier in oogenesis; it interacts specifically with the DDX6 helicase Xp54; it is detected in polysomes and stalled translation initiation complexes; its over-expression leads to selective binding to translatable mRNA species without evidence of translation repression or mRNA degradation. Since both Xp54 and xRAPA are repressors of translation, activation appears to be effected through targeting of xRAPB/Xp54.

Musashi Protein-directed Translational Activation of Target mRNAs Is Mediated by the Poly(A) Polymerase, Germ Line Development Defective-2

The mRNA-binding protein, Musashi, has been shown to regulate translation of select mRNAs and to control cellular identity in both stem cells and cancer cells. Within the mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed Musashi's capability to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly(A) polymerase germ line development defective-2 (GLD2) and map the association domain to 31 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target 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.

Zar1 represses translation in Xenopus oocytes and binds to the TCS in maternal mRNAs with different characteristics than Zar2

Maternal mRNAs are translationally regulated during early development. Zar1 and its closely related homolog, Zar2, are both crucial in early development. Xenopus laevis Zygote arrest 2 (Zar2) binds to the Translational Control Sequence (TCS) in maternal mRNAs and regulates translation. The molecular mechanism of Zar1 has not been described. Here we report similarities and differences between Xenopus Zar1 and Zar2. Analysis of Zar sequences in vertebrates revealed two Zar family members with conserved, characteristic amino acid differences in the C-terminal domain. The presence of only two vertebrate Zar proteins was supported by analyzing Zar1 synteny. We propose that the criteria for naming Zar sequences are based on the characteristic amino acids and the chromosomal context. We also propose reclassification of some Zar sequences. We found that Zar1 is expressed throughout oogenesis and is stable during oocyte maturation. The N-terminal domain of Zar1 repressed translation of a reporter construct in immature oocytes. Both Zar1 and Zar2 bound to the TCS in the Wee1 and Mos 3′ UTRs using a zinc finger in the C-terminal domain. However, Zar1 had much higher affinity for RNA than Zar2. To show the functional significance of the conserved amino acid substitutions, these residues in Zar2 were mutated to those found in Zar1. We show that these residues contributed to the different RNA binding characteristics of Zar1 compared to Zar2. Our study shows that Zar proteins have generally similar molecular functions in the translational regulation of maternal mRNAs, but they may have different roles in early development.

Xenopus laevis zygote arrest 2 (zar2) encodes a zinc finger RNA-binding protein that binds to the translational control sequence in the maternal Wee1 mRNA and regulates translation

Zygote arrest (Zar) proteins are crucial for early embryonic development, but their molecular mechanism of action is unknown. The Translational Control Sequence (TCS) in the 3′ untranslated region (UTR) of the maternal mRNA, Wee1, mediates translational repression in immature Xenopus oocytes and translational activation in mature oocytes, but the protein that binds to the TCS and mediates translational control is not known. Here we show that Xenopus laevis Zar2 (encoded by zar2) binds to the TCS in maternal Wee1 mRNA and represses translation in immature oocytes. Using yeast 3 hybrid assays and electrophoretic mobility shift assays, Zar2 was shown to bind specifically to the TCS in the Wee1 3′UTR. RNA binding required the presence of Zn2+ and conserved cysteines in the C-terminal domain, suggesting that Zar2 contains a zinc finger. Consistent with regulating maternal mRNAs, Zar2 was present throughout oogenesis, and endogenous Zar2 co-immunoprecipitated endogenous Wee1 mRNA from immature oocytes, demonstrating the physiological significance of the protein–RNA interaction. Interestingly, Zar2 levels decreased during oocyte maturation. Dual luciferase reporter tethered assays showed that Zar2 repressed translation in immature oocytes. Translational repression was relieved during oocyte maturation and this coincided with degradation of Zar2 during maturation. This is the first report of a molecular function of zygote arrest proteins. These data show that Zar2 contains a zinc finger and is a trans-acting factor for the TCS in maternal mRNAs in immature Xenopus oocytes.

Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes

Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine122 is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.

Dominant-Negative, Spliced Variant of the Intermediate-Conductance Ca2+-Activated K+ Channel, KCa3.1 in Lymphoid Cells

Intermediate-conductance Ca2+-activated K+ channel (IKCa channel) encoded by KCa3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of KCa3.1 (hKCa3.1b) from the human thymus, which were lacking the N-terminal domains of the original hKCa3.1a as a result of alternative splicing. hKCa3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hKCa3.1a proteins were mainly expressed in plasma membrane fraction, whereas hKCa3.1b was in the cytoplasmic fraction. We also identified a similar N-terminus lacking KCa3.1 spliced variants from mice and rat lymphoid tissues (mKCa3.1b, rKCa3.1b). In the HEK293 heterologous expression system, the cellular distribution of CFP-tagged hKCa3.1a and/or YFP-tagged hKCa3.1b isoforms showed that hKCa3.1b suppressed the localization of hKCa3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hKCa3.1b with hKCa3.1a suppressed IKCa channel activity of hKCa3.1a in a dominant-negative manner. In addition, the present study indicated that up-regulation of mKCa3.1b in mice thymocytes differentiated CD4+CD8+ phenotype thymocytes into CD4-CD8- ones, and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mKCa3.1b over-expressing mice thymocytes. These suggest that the N-terminal domain of KCa3.1 is critical for channel trafficking to the plasma membrane, and that the fine tuning of IKCa channel activity modulated through alternative splicing may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

Involvement of Dominant-negative Spliced Variants of the Intermediate Conductance Ca2+-activated K+ Channel, KCa3.1, in Immune Function of Lymphoid Cells

The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

Smaug assembles an ATP-dependent stable complex repressingnanosmRNA translation at multiple levels

The nanos (nos) mRNA encodes the posterior determinant of the Drosophila embryo. Translation of the RNA is repressed throughout most of the embryo by the protein Smaug binding to Smaug recognition elements (SREs) in the 3' UTR. Translation is locally activated at the posterior pole by Oskar. This paper reports that the SREs govern the time- and ATP-dependent assembly of an exceedingly stable repressed ribonucleoprotein particle (RNP) in embryo extract. Repression can be virtually complete. Smaug and its co-repressor Cup as well as Trailer hitch and the DEAD box protein Me31B are part of the repressed RNP. The initiation factor eIF4G is specifically displaced, and 48S pre-initiation complex formation is inhibited. However, later steps in translation initiation are also sensitive to SRE-dependent inhibition. These data confirm several previously untested predictions of a current model for Cup-dependent repression but also suggest that the Cup model by itself is insufficient to explain translational repression of the nos RNA. In the embryo extract, recombinant Oskar relieves translational repression and deadenylation by preventing Smaug's binding to the SREs.

Transgenic zebrafish reveals novel mechanisms of translational control of cyclin B1 mRNA in oocytes

Temporal translation control of localized mRNA is crucial for regulating various cellular and developmental processes. However, little is known about the mechanisms of temporal translation control of localized mRNA due to the limitation in technology. cyclin B1 mRNA at the animal polar cytoplasm of immature zebrafish oocytes is translationally repressed, and its activation is temporally regulated during maturation. Mechanisms of cyclin B1 translation in oocytes were analyzed using transgenic zebrafish in which reporter mRNAs are produced from transgenes introduced into the genome through transcription in the nucleus followed by transport to the cytoplasm, as in endogenous mRNAs. Real-time imaging of the site and timing of translation showed that mRNAs containing the full-length cyclin B1 sequence precisely mimic the localization and translation of endogenous cyclin B1 mRNA. However, mRNAs containing cyclin B1 3′ untranslated region but lacking open reading frame (ORF) underwent abnormal localization and precocious translational activation, indicating the significance of the ORF in translational control of cyclin B1 mRNA. Our genetic approach in combination with real-time imaging of the translation site and timing provides a novel insight into the mechanisms of temporal control of translation.

Distinct functions of maternal and somatic Pat1 protein paralogs.

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5' UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.

Zif-1 translational repression defines a second, mutually exclusive OMA function in germline transcriptional repression.

Specification of primordial germ cells requires global repression of transcription. In C. elegans, primordial germ cells are generated through four rounds of asymmetric divisions, starting from the zygote P0, each producing a transcriptionally repressed germline blastomere (P1-P4). Repression in P2-P4 requires PIE-1, which is provided maternally in oocytes and segregated to all germline blastomeres. We have shown previously that OMA-1 and OMA-2 repress global transcription in P0 and P1 by sequestering TAF-4, an essential component of TFIID. Soon after the first mitotic cycle, OMA proteins undergo developmentally regulated degradation. Here, we show that OMA proteins also repress transcription in P2-P4 indirectly, through a completely different mechanism that operates in oocytes. OMA proteins bind to both the 3' UTR of the zif-1 transcript and the eIF4E-binding protein, SPN-2, repressing translation of zif-1 mRNA in oocytes. zif-1 encodes the substrate-binding subunit of the E3 ligase for PIE-1 degradation. Inhibition of zif-1 translation in oocytes ensures high PIE-1 levels in oocytes and germline blastomeres. The two OMA protein functions are strictly regulated in both space and time by MBK-2, a kinase activated following fertilization. Phosphorylation by MBK-2 facilitates the binding of OMA proteins to TAF-4 and simultaneously inactivates their function in repressing zif-1 translation. Phosphorylation of OMA proteins displaces SPN-2 from the zif-1 3' UTR, releasing translational repression. We propose that MBK-2 phosphorylation serves as a developmental switch, converting OMA proteins from specific translational repressors in oocytes to global transcriptional repressors in embryos, together effectively repressing transcription in all germline blastomeres.

Porcine CPEB1 is involved in Cyclin B translation and meiotic resumption in porcine oocytes

Ovarian immature oocytes accumulate many dormant maternal mRNAs, which have short poly(A) tails. Cytoplasmic-polyadenylation-element binding protein (CPEB) has been reported to play key roles for the elongation of the tails and the translation of these mRNAs in Xenopus oocytes. However, the functions of CPEB in meiotic resumption have not yet been established in mammalian oocytes. The present study examined the roles of porcine CPEB in Cyclin B syntheses and meiotic resumption of porcine oocytes. Porcine CPEB1 (pCPEB1) cDNA was cloned from total RNA of immature oocytes by RT-PCR. The overexpression of pCPEB1 by mRNA injection into immature oocytes increased Cyclin B expression and the rate of meiotic resumption. Conversely, the inhibition of endogenous CPEB by expression of a dominant-negative mutant pCPEB1 (AA-CPEB), which replaced the expected phosphorylation sites with alanines, had the effect of inhibiting Cyclin B synthesis, ribosomal S6 kinase phosphorylation (an indicator of Mos activity), and meiotic resumption. The inhibition of porcine Aurora A by an injection of antisense RNA enhanced the inhibitory effects of AA-CPEB. These results suggest the involvement of mammalian CPEB1 in Cyclin B syntheses and meiotic resumption in mammalian oocytes. In addition, the phosphorylation sites of pCPEB1 were identified and are suggested to be phosphorylated by porcine Aurora A.

Oocyte maturation failure: a syndrome of bad eggs

To show that disruption of meiotic competence results in cell cycle arrest, and the production of immature oocytes that are not capable of fertilization. Through an extensive review of animal studies and clinical case reports, we define the syndrome of oocyte maturation failure as a distinct oocyte disorder, present a classification system based on clinical parameters, and discuss the potential molecular origins for the disease.

Polyribosome analysis for investigating mRNA translation in Xenopus oocytes, eggs and embryos

The earliest stages of animal development occur without the benefit of zygotic transcription. The absence of transcription necessitates that all changes in the levels of specific proteins must be controlled by post-transcriptional mechanisms, such as the regulated translation of stored maternal mRNAs. One of the major challenges to investigating translational mechanisms is the availability of reliable methods for assaying the translational state of specific mRNAs. The most definitive assay of an mRNA’s translational state is polyribosome association; mRNAs actively translated are engaged with polyribosomes while mRNAs translationally repressed are not. While linear gradient centrifugation is commonly used to purify polyribosomes from a wide variety of cell types in different organisms, the isolation of polyribosomes from Xenopus oocytes, eggs and embryos presents some unique challenges. Here we detail the methodology for the isolation and analysis of polyribosomes from Xenopus oocytes, eggs and embryos using step gradient centrifugation. We present detailed protocols, describe the critical controls and provide several examples to guide the interpretation of experimental results regarding the translational state of specific mRNAs.

Pumilio 2 controls translation by competing with eIF4E for 7-methyl guanosine cap recognition

Pumilio 2 (Pum2) interacts with the 3' UTR-containing pumilio binding element (PBE) of RINGO/SPY mRNA to repress translation in Xenopus oocytes. Here, we show that Pum2 also binds directly to the 5' 7mG cap structure; in so doing, it precludes eIF4E from binding the cap. Using deletion analysis, we have mapped the cap interaction domain of Pum2 to the amino terminus of the protein and identified a conserved tryptophan residue that mediates this specific interaction. Reporter mRNA-based assays demonstrate that Pum2 requires the conserved tryptophan to repress translation in injected Xenopus oocytes. Thus, in addition to its suggested role in regulating poly(A) tail length and mRNA stability, our results suggest that vertebrate Pumilio can repress translation by blocking the assembly of the essential initiation complex on the cap.

In Vivo Study on Vertical Transmission of the HIV-1 gag Gene via Mouse Oocytes

Acquired immunodeficiency syndrome (AIDS) is a major public health problem worldwide. This study was performed to explore the feasibility of vertical transmission of human immunodeficiency virus-1 (HIV-1) gag gene via oocyte. The recombinant plasmid (pIRES2-EGFP-gag) was injected into mouse ovaries to transfect germ cells. Induction of superovulation and then animal mating were performed to collect oocytes and two-cell embryos. Positive FISH signals for HIV-1 gag DNA were detected in the nuclei of oocytes and embryos, and in chromosomes of mature oocytes, indicated integration of the gene into the oocyte genome and gene replication in the embryo. HIV-1 gag cDNA positive bands detected by RT-PCR in oocytes and embryos indicated successful gene transcription, while positive immunofluorescence signals for HIV-1 gag protein indicated successful translation in both oocytes and embryos. The HIV-1 gag gene was transmitted vertically to the next generation via oocytes and it retained its function in replication, transcription and translation following at least one mitotic division in embryos.

174 MicroRNA EXPRESSION PROFILE IN BOVINE CUMULUS-OOCYTE COMPLEXES DURING LATE OOGENESIS

During late oogenesis, the mammalian oocyte synthesizes and stores mRNA necessary to guide the early stages of embryo development before the activation of embryonic transcription. The oocyte also contains many post-transcriptional regulatory mechanisms that coordinate mRNA stability and translation before specific activation. MicroRNAs (miRNAs) are short noncoding RNAs (17–25 nucleotides) that repress translation of target genes through sequence complementation and have recently been identified in murine oocytes. The objective of the current study was to identify and characterize the expression of miRNAs in bovine cumulus–oocyte complexes (COC) during late oogenesis as a potential mechanism for post-transcriptional regulation of mRNA in developing bovine oocytes. Ovaries from beef cattle (mixed populations) were obtained at a local abattoir. The COC were aspirated from 2- to 10-mm follicles and were pooled from each of 5 replicate collections for RNA extraction (n = 2241 total COC). Small RNA in the 16- to 27-bp range was isolated and used to construct cDNA libraries for sequencing, producing 2529 successful sequences that were clustered based on matching 14 consecutive bases to the most common member of the cluster. The consensus sequences of the clusters were screened for mitochondrial RNA, rRNA, tRNA, and snoRNA contaminants, leading to removal of 774 (31%) sequences from consideration. The remaining 1755 putative miRNA sequences were compared with known miRNA in miRBase, revealing 62 bovine COC miRNA clusters matching previously known sequences and 4 with no match. The cluster with the largest number of sequences identified in bovine COC matched the sequence of the let-7 miRNA family (657 sequences or 37% of putative miRNA). Within the let-7 family, let-7b (459 sequences or 26%) was the most abundant followed by let-7i (135 sequences or 8%). The four clusters that did not match sequences in miRBase represent putative novel miRNA. One of these four clusters had relatively high expression in bovine COCs (308 sequences or 18%), whereas the other 3 clusters had relatively low expression (total of 55 combined sequences or 3%). Expression of several putative miRNAs (let-7b, let-7i, miR-106a, and the abundant novel miRNA) in bovine COC were confirmed using TaqMan miRNA assays. These results demonstrate the presence of miRNA within bovine COC during late oogenesis, which suggests that these post-transcriptional regulatory elements may play a role in coordinating mRNA stability and translation in bovine oocytes.

CPEB Interacts with an Ovary-specific eIF4E and 4E-T in Early Xenopus Oocytes

CPEB (cytoplasmic polyadenylation element-binding protein) is an important regulator of translation in oocytes and neurons. Although previous studies of CPEB in late Xenopus oocytes involve the eIF4E-binding protein maskin as the key factor for the repression of maternal mRNA, a second mechanism must exist, since maskin is absent earlier in oogenesis. Using co-immunoprecipitation and gel filtration assays, we show that CPEB specifically interacts, via protein/protein interactions, with the RNA helicase Xp54, the RNA-binding proteins P100(Pat1) and RAP55, the eIF4E-binding protein 4E-T, and an eIF4E protein. Remarkably, these CPEB complex proteins have been characterized, in one or more organism, as P-body, maternal, or neuronal granule components. We do not detect interactions with eIF4E1a, the canonical cap-binding factor, eIF4G, or eIF4A or with proteins expressed late in oogenesis, including maskin, PARN, and 4E-BP1. The eIF4E protein was identified as eIF4E1b, a close homolog of eIF4E1a, whose expression is restricted to oocytes and early embryos. Although eIF4E1b possesses all residues required for cap and eIF4G binding, it binds m(7)GTP weakly, and in pull-down assays, rather than binding eIF4G, it binds 4E-T, in a manner independent of the consensus eIF4E-binding site, YSKEELL. Wild type and Y-A mutant 4E-T (which binds eIF4E1b but not eIF4E1a), when tethered to a reporter mRNA, represses its translation in a cap-dependent manner, and injection of eIF4E1b antibody accelerates meiotic maturation. Altogether, our data suggest that CPEB, partnered with several highly conserved RNA-binding partners, inhibits protein synthesis in oocytes using a novel pairing of 4E-T and eIF4E1b.

RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

One activity that controls mRNA translation in vertebrate oocytes, embryos, and neurons is cytoplasmic polyadenylation. In Xenopus oocytes, where much of the biochemistry of this process has been elucidated, nuclear pre-mRNAs containing a cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) have long poly(A) tails; once the RNAs are spliced and transported to the cytoplasm, the tails are shortened. Following the resumption of meiosis, the poly(A) tails are lengthened and translation ensues. CPEB is a sequence-specific RNA-binding protein that coordinates these events and does so by binding to the CPE as well as several factors including Gld2, a poly(A) polymerase, and PARN [poly(A)-specific ribonuclease], a deadenylase. Here, we show that ePAB, embryonic poly(A)-binding protein, transiently associates with the polyadenylation complex; it initially interacts with CPEB, but after polyadenylation, it binds the poly(A) tail. ePAB dissociation from CPEB is regulated by RINGO (Rapid Inducer of G(2)/M progression in Oocytes), a cyclin B1-like cofactor that activates cdk1, a protein kinase that phosphorylates CPEB. Subsequent ePAB binding to the poly(A) tail is necessary to protect the homopolymer from degradation by deadenylating enzymes. Poly(A)-bound ePAB also interacts with eIF4G, which instigates translation initiation of CPEB-bound mRNAs.