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The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo Caenorhabditis elegans oogenesis model to characterize the properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and three other translational regulators. We demonstrate that MEX-3 undergoes phase separation and appears to have intrinsic gel-like properties in vitro. We also identify novel roles for the chaperonin-containing tailless complex polypeptide 1 (CCT) chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. The CCT chaperonin and actin also oppose the expansion of endoplasmic reticulum sheets that may promote ectopic condensation of RNA-binding proteins. These novel regulators of condensation are also required for the translational repression of maternal mRNA which is essential for oocyte quality and fertility. The identification of this regulatory network may also have implications for understanding the role of hMex3 phase transitions in cancer., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Oocyte in vitro maturation is a technique in assisted reproductive technology. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (MII) oocytes compared to those matured in vivo in bovines, mice, and humans. The mechanisms underlying this phenomenon are poorly understood. Here, we use poly(A) inclusive RNA isoform sequencing (PAIso-seq) for profiling the transcriptome-wide poly(A) tails in both in vivo and in vitro matured mouse and human oocytes. Our results demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes. Moreover, the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes, contributing to reduced translation of these deadenylase machinery components and subsequently impaired global maternal mRNA deadenylation. Our findings highlight impaired maternal mRNA deadenylation as a distinct molecular defect in in vitro MII oocytes., (© 2024. The Author(s).)

Rapid cleavage divisions and the transition from maternal to zygotic control of gene expression are the hallmarks of early embryonic development in most species. Early development in insects, fish and amphibians is characterized by several short cell cycles with no gap phases, necessary for the rapid production of cells prior to patterning and morphogenesis. Maternal mRNAs and proteins loaded into the egg during oogenesis are essential to drive these rapid early divisions. Once the function of these maternal inputs is complete, the maternal-to-zygotic transition (MZT) marks the handover of developmental control to the gene products synthesized from the zygotic genome. The MZT requires three major events: the removal of a subset of maternal mRNAs, the initiation of zygotic transcription, and the remodeling of the cell cycle. In each species, the MZT occurs at a highly reproducible time during development due to a series of feedback mechanisms that tightly couple these three processes. Dissecting these feedback mechanisms and their spatiotemporal control will be essential to understanding the control of the MZT. In this primer, we outline the mechanisms that govern the major events of the MZT across species and highlight the role of feedback mechanisms that ensure the MZT is precisely timed and orchestrated., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The transition from oocyte to embryo requires translation of maternally provided transcripts that in Drosophila is activated by Pan Gu kinase to release a rapid succession of 13 mitotic cycles. Mitotic entry is promoted by several protein kinases that include Greatwall/Mastl, whose Endosulfine substrates antagonize Protein Phosphatase 2A (PP2A), facilitating mitotic Cyclin-dependent kinase 1/Cyclin B kinase activity. Here we show that hyperactive greatwall Scant can not only be suppressed by mutants in its Endos substrate but also by mutants in Pan Gu kinase subunits. Conversely, mutants in me31B or trailer hitch, which encode a complex that represses hundreds of maternal mRNAs, enhance greatwall Scant . Me31B and Trailer Hitch proteins, known substrates of Pan Gu kinase, copurify with Endos. This echoes findings that budding yeast Dhh1, orthologue of Me31B, associates with Igo1/2, orthologues of Endos and substrates of the Rim15, orthologue of Greatwall. endos- derived mutant embryos show reduced Me31B and elevated transcripts for the mitotic activators Cyclin B, Polo and Twine/Cdc25. Together, our findings demonstrate a previously unappreciated conservation of the Greatwall-Endosulfine pathway in regulating translational repressors and its interactions with the Pan Gu kinase pathway to regulate translation and/or stability of maternal mRNAs upon egg activation.

Obesity has significant repercussions for female reproductive health, including adverse effects on oocyte quality, fertility, embryo development and offspring health. Here, we showed that intermittent fasting (IF) has several notable effects on follicular development, oocyte development and maturation and offspring health in obese mice. IF treatment prevents obesity-associated germline-soma communication defects, mitochondrial dysfunction, oxidative damage, apoptosis, and spindle/chromosomal disruption. RNA-sequencing analysis of oocytes from normal diet (ND), high-fat diet (HFD), and HFD + IF mice indicated that IF treatment improved mitochondrial oxidative phosphorylation function and mRNA storage and translation, which was potentially mediated by the Smith-like family member 14 B (LSM14B). Knockdown of LSM14B by siRNA injection in oocytes from ND mice recapitulates all the translation, mitochondrial dysfunction and meiotic defect phenotypes of oocytes from HFD mice. Remarkably, the injection of Lsm14b mRNA into oocytes from HFD mice rescued the translation, mitochondrial dysfunction and meiotic defect phenotypes. These results demonstrated that dysfunction in the oocyte translation program is associated with obesity-induced meiotic defects, while IF treatment increased LSM14B expression and maternal mRNA translation and restored oocyte quality. This research has important implications for understanding the effects of obesity on female reproductive health and offers a potential nonpharmacological intervention to improve oocyte quality and fertility in obese individuals., Competing Interests: Declaration of competing interest All of the contributing authors declared no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A large number of mRNAs of maternal origin are produced during oogenesis and deposited in the oocyte. Since transcription stops at the onset of meiosis during oogenesis and does not resume until later in embryogenesis, maternal mRNAs are the only templates for protein synthesis during this period. To ensure that a protein is made in the right place at the right time, the translation of maternal mRNAs must be activated at a specific stage of development. Here we summarize our current understanding of the sophisticated mechanisms that contribute to the temporal repression of maternal mRNAs, termed maternal mRNA dormancy. We discuss mechanisms at the level of the RNA itself, such as the regulation of polyadenine tail length and RNA modifications, as well as at the level of RNA-binding proteins, which often block the assembly of translation initiation complexes at the 5' end of an mRNA or recruit mRNAs to specific subcellular compartments. We also review microRNAs and other mechanisms that contribute to repressing translation, such as ribosome dormancy. Importantly, the mechanisms responsible for mRNA dormancy during the oocyte-to-embryo transition are also relevant to cellular quiescence in other biological contexts., (© 2024 The Author(s).)

Maternal mRNAs accumulate during egg growth and must be judiciously degraded or translated to ensure successful development of mammalian embryos. In this review we integrate recent investigations into pathways controlling rapid degradation of maternal mRNAs during the maternal-to-zygotic transition. Degradation is not indiscriminate, and some mRNAs are selectively protected and rapidly translated after fertilization for reprogramming the zygotic genome during early embryogenesis. Oocyte specific cofactors and pathways have been illustrated to control different futures of maternal mRNAs. We discuss mechanisms that control the fate of maternal mRNAs during late oogenesis and after fertilization. Issues to be resolved in current maternal mRNA research are described, and future research directions are proposed., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Ltd.)

Poly(A)-binding proteins (PABPs) play roles in mRNA maturation, translational activity, and decay. The functions of PABPs, especially PABPN1 and PABPC1, in somatic cells have been well-studied. However, little is known about the roles of PABPs in oocytes because of the unique mechanisms of mRNA metabolism in oocytes. This study focused on PABPN1L and generated Pabpn1l knockout (KO) mice using the CRISPR/Cas9 system. After mating tests, we found that Pabpn1l KO females were infertile due to the failure of the embryos to develop to the 4-cell stage. RNA-seq analysis revealed aberrant mRNA persistence in Pabpn1l KO-MII oocytes, which indicates impaired mRNA degradation during the germinal vesicle (GV) to MII transition. We also revealed that the exogenous expression of Pabpn1l mRNA in KO-GV oocytes recovered defects of embryonic development. PABPN1L is partly indispensable for female fertility in mice, owing to its necessity for embryonic development, which is supported by mRNA degradation during GV to MII maturation.

Transcriptional and post-transcriptional regulations control gene expression in most cells. However, critical transitions during the development of the female gamete relies exclusively on regulation of mRNA translation in the absence of de novo mRNA synthesis. Specific temporal patterns of maternal mRNA translation are essential for the oocyte progression through meiosis, for generation of a haploid gamete ready for fertilization and for embryo development. In this review, we will discuss how mRNAs are translated during oocyte growth and maturation using mostly a genome-wide perspective. This broad view on how translation is regulated reveals multiple divergent translational control mechanisms required to coordinate protein synthesis with progression through the meiotic cell cycle and with development of a totipotent zygote., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Oocyte maturation is accompanied by changes in abundances of thousands of mRNAs, many degraded and many preferentially stabilized. mRNA stability can be regulated by diverse features including GC content, codon bias, and motifs within the 3'-untranslated region (UTR) interacting with RNA binding proteins (RBPs) and miRNAs. Many studies have identified factors participating in mRNA splicing, bulk mRNA storage, and translational recruitment in mammalian oocytes, but the roles of potentially hundreds of expressed factors, how they regulate cohorts of thousands of mRNAs, and to what extent their functions are conserved across species has not been determined. We performed an extensive in silico cross-species analysis of features associated with mRNAs of different stability classes during oocyte maturation (stable, moderately degraded, and highly degraded) for five mammalian species. Using publicly available RNA sequencing data for germinal vesicle (GV) and MII oocyte transcriptomes, we determined that 3'-UTR length and synonymous codon usage are positively associated with stability, while greater GC content is negatively associated with stability. By applying machine learning and feature selection strategies, we identified RBPs and miRNAs that are predictive of mRNA stability, including some across multiple species and others more species-restricted. The results provide new insight into the mechanisms regulating maternal mRNA stabilization or degradation. NEW & NOTEWORTHY Conservation across species of mRNA features regulating maternal mRNA stability during mammalian oocyte maturation was analyzed. 3'-Untranslated region length and synonymous codon usage are positively associated with stability, while GC content is negatively associated. Just three RNA binding protein motifs were predicted to regulate mRNA stability across all five species examined, but associated pathways and functions are shared, indicating oocytes of different species arrive at comparable physiological destinations via different routes.

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report that the germline-specific eIF4E paralog, eIF4E1b, is essential for zebrafish oogenesis. eIF4E1b localizes to P-bodies in zebrafish embryos and binds to mRNAs with reported short or no polyA tails, including histone mRNAs. Loss of eIF4E1b results in reduced histone mRNA levels in early gonads, consistent with a role in mRNA storage. Using mouse and human eIF4E1Bs (in vitro) and zebrafish eIF4E1b (in vivo), we show that unlike canonical eIF4Es, eIF4E1b does not interact with eIF4G to initiate translation. Instead, eIF4E1b interacts with the translational repressor eIF4ENIF1, which is required for eIF4E1b localization to P-bodies. Our study is consistent with an important role of eIF4E1b in regulating mRNA dormancy and provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development., (© 2023. The Author(s).)

Postovulatory aging leads to the decline in oocyte quality and subsequent impairment of embryonic development, thereby reducing the success rate of assisted reproductive technology (ART). Potential preventative strategies preventing oocytes from aging and the associated underlying mechanisms warrant investigation. In this study, we identified that cordycepin, a natural nucleoside analogue, promoted the quality of oocytes aging in vitro, as indicated by reduced oocyte fragmentation, improved spindle/chromosomes morphology and mitochondrial function, as well as increased embryonic developmental competence. Proteomic and RNA sequencing analyses revealed that cordycepin inhibited the degradation of several crucial maternal proteins and mRNAs caused by aging. Strikingly, cordycepin was found to suppress the elevation of DCP1A protein by inhibiting polyadenylation during postovulatory aging, consequently impeding the decapping of maternal mRNAs. In humans, the increased degradation of DCP1A and total mRNA during postovulatory aging was also inhibited by cordycepin. Collectively, our findings demonstrate that cordycepin prevents postovulatory aging of mammalian oocytes by inhibition of maternal mRNAs degradation via suppressing polyadenylation of DCP1A mRNA, thereby promoting oocyte developmental competence., (© 2023. The Author(s).)

Control of mRNA poly(A) tails is essential for regulation of mRNA metabolism, specifically translation efficiency and mRNA stability. Gene expression in maturing oocytes relies largely on post-transcriptional regulation, as genes are transcriptionally silent during oocyte maturation. The CCR4-NOT complex is a major mammalian deadenylase, which regulates poly(A) tails of maternal mRNAs; however, the function of the CCR4-NOT complex in translational regulation has not been well understood. Here, we show that this complex suppresses translational activity of maternal mRNAs during oocyte maturation. Oocytes lacking all CCR4-NOT deadenylase activity owing to genetic deletion of its catalytic subunits, Cnot7 and Cnot8, showed a large-scale gene expression change caused by increased translational activity during oocyte maturation. Developmental arrest during meiosis I in these oocytes resulted in sterility of oocyte-specific Cnot7 and Cnot8 knockout female mice. We further showed that recruitment of CCR4-NOT to maternal mRNAs is mediated by the 3'UTR element CPE, which suppresses translational activation of maternal mRNAs. We propose that suppression of untimely translational activation of maternal mRNAs via deadenylation by CCR4-NOT is essential for proper oocyte maturation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The accumulation and storage of maternal mRNA is crucial for oocyte maturation and embryonic development. PATL2 is an oocyte-specific RNA-binding protein, and previous studies have confirmed that PATL2 mutation in humans and knockout mice cause oocyte maturation arrest or embryonic development arrest, respectively. However, the physiological function of PATL2 in the process of oocyte maturation and embryonic development is largely unknown. Here, we report that PATL2 is highly expressed in growing oocytes and couples with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The germinal vesicle oocytes from Patl2-/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed that PATL2 phosphorylation occurs in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found that the S279D mutation decreased the protein level of PATL2 and led to subfertility in Palt2S279D knock-in mice. Our work reveals the previously unrecognized role of PATL2 in regulating the maternal transcriptome and shows that phosphorylation of PATL2 leads to the regulation of PATL2 protein levels via ubiquitin-mediated proteasomal degradation in oocytes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Vertebrate oocytes face a particular challenge concerning the regulation of gene expression during meiotic maturation. Global transcription becomes quiescent in fully grown oocytes, remains halted throughout maturation and fertilization, and only resumes upon embryonic genome activation. Hence, the oocyte meiotic maturation process is largely regulated by protein synthesis from pre-existing maternal messenger RNAs (mRNAs) that are transcribed and stored during oocyte growth. Rapidly developing genome-wide techniques have greatly expanded our insights into the global translation changes and possible regulatory mechanisms during oocyte maturation. The storage, translation, and processing of maternal mRNAs are thought to be regulated by factors interacting with elements in the mRNA molecules. Additionally, posttranscriptional modifications of mRNAs, such as methylation and uridylation, have recently been demonstrated to play crucial roles in maternal mRNA destabilization. However, a comprehensive understanding of the machineries that regulate maternal mRNA fate during oocyte maturation is still lacking. In particular, how the transcripts of important cell cycle components are stabilized, recruited at the appropriate time for translation, and eliminated to modulate oocyte meiotic progression remains unclear. A better understanding of these mechanisms will provide invaluable insights for the preconditions of developmental competence acquisition, with important implications for the treatment of infertility. This review discusses how the storage, localization, translation, and processing of oocyte mRNAs are regulated, and how these contribute to oocyte maturation progression., (© 2023 Cambridge Philosophical Society.)

Mammalian oogenesis features reliance on the mRNAs produced and stored during early growth phase. These are essential for producing an oocyte competent to undergo meiotic maturation and embryogenesis later when oocytes are transcriptionally silent. The fate of maternal mRNAs hence ensures the success of oogenesis and the quality of the resulting eggs. Nevertheless, how the fate of maternal mRNAs is determined remains largely elusive. RNA-binding proteins (RBPs) are crucial regulators of oogenesis, yet the identity of the full complement of RBPs expressed in oocytes is unknown. Here, a global view of oocyte-expressed RBPs is presented: mRNA-interactome capture identifies 1396 RBPs in mouse oocytes. An analysis of one of these RBPs, LSM family member 14 (LSM14B), demonstrates that this RBP is specific to oocytes and associated with many networks essential for oogenesis. Deletion of Lsm14b results in female-specific infertility and a phenotype characterized by oocytes incompetent to complete meiosis and early embryogenesis. LSM14B serves as an interaction hub for proteins and mRNAs throughout oocyte development and regulates translation of a subset of its bound mRNAs. Therefore, RNP complexes tethered by LSM14B are found exclusively in oocytes and are essential for the control of maternal mRNA fate and oocyte development., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Strategies to maximize individual fertility chances are constant requirements of ART. In vitro folliculogenesis may represent a valid option to create a large source of immature ovarian follicles in ART. Efforts are being made to set up mammalian follicle culture protocols with suitable FSH stimuli. In this study, a new type of recombinant FSH (KN015) with a prolonged half-life is proposed as an alternative to canonical FSH. KN015 supports the in vitro development of mouse follicles from primary to preovulatory stage with higher efficiency than canonical FSH and enhanced post-fertilization development rates of the ovulated oocytes. The use of KN015 also allows us to compare the dynamic transcriptome changes in oocytes and granulosa cells at different stages, in vivo and in vitro. In particular, KN015 facilitates mRNA accumulation in growing mouse oocytes and prevents spontaneous luteinization of granulosa cells in vitro. Novel analyses of transcriptome changes in this study reveal that the in vivo oocytes were more efficient than in vitro oocytes in terms of maternal mRNA clearing during meiotic maturation. KN015 promotes the degradation of maternal mRNA during in vitro oocyte maturation, improves cytoplasmic maturation and, therefore, enhances embryonic developmental potential. These findings establish new transcriptome data for oocyte and granulosa cells at the key stages of follicle development, and should help to widen the use of KN015 as a valid and commercially available hormonal support enabling optimized in vitro development of follicles and oocytes., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Most of the resources that support the early development of the embryo are stored in the oocyte. Clearing of maternal resources and activation of the embryonic genome to produce its own mRNA transcripts marks the maternal-to-embryo transition. Dependence on stored mRNA can last from a few hours to several days, depending on animal species. The mechanisms regulating stabilization and recruitment of stored maternal transcripts have not yet been described in full detail but are known to involve reversible polyadenylation and modulation of 3'UTR-mediated elements. RNA epigenetic modifications, new players in this field, have an important role in RNA regulation and stabilization., Results: The objectives of this study were first to determine if some of post-transcriptional methylation of stored mRNA is greater in oocytes than in somatic cells. We found that m 6 A, known to be the most prevalent and involved in various aspects of RNA metabolism and physiological functions, is particularly abundant in porcine oocyte mRNA compared to liver used as a somatic tissue reference. The second objective was to compare the epitranscriptome machinery, such as methyltransferases ("writers"), binding proteins ("readers") and demethylases ("erasers") catalyzing the different process, in follicles and oocytes of different mammalian species by immunofluorescence and confocal microscopy. The expression and localization patterns of these proteins differ between mice, pigs and cows ovaries and oocytes. m 5 C-associated proteins were generally less abundant. In contrast, m 6 A-associated proteins were expressed strongly during the early and late stages of folliculogenesis. Transzonal projections were found to contain more granules bearing the m 5 C mark in mice but both m 5 C and m 6 A methylation marks in association with mature oocytes of pigs and cows. Eraser proteins showed the greatest interspecies diversity in terms of distribution in the germinal tissues., Conclusions: So far, few studies have looked at the oocyte and ovarian epitranscriptomic profile. Our findings indicate that a hitherto unrecognized species-specific layer of transcript regulation occurs at the RNA level and might be consequential during the oocyte transcriptional silencing period., (© 2023. The Author(s).)

Translation of maternal mRNAs is detected before transcription of zygotic genes and is essential for mammalian embryo development. How certain maternal mRNAs are selected for translation instead of degradation and how this burst of translation affects zygotic genome activation remain unknown. Using gene-edited mice, we document that the oocyte-specific eukaryotic translation initiation factor 4E family member 1b (eIF4E1b) is the regulator of maternal mRNA expression that ensures subsequent reprogramming of the zygotic genome. In oocytes, eIF4E1b binds to transcripts encoding translation machinery proteins, chromatin remodelers, and reprogramming factors to promote their translation in zygotes and protect them from degradation. The protein products are thought to establish an open chromatin landscape in one-cell zygotes to enable transcription of genes required for cleavage stage development. Our results define a program for rapid resetting of the zygotic epigenome that is regulated by maternal mRNA expression and provide new insights into the mammalian maternal-to-zygotic transition., (Published by Cold Spring Harbor Laboratory Press.)

Background: Studies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown., Methods: Female C57BL/6 mice (6-8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription-quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions., Results: Embryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor., Conclusions: Sperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos., (© 2023. The Author(s).)

Maternal messenger ribonucleic acids (mRNAs) are driven by a highly orchestrated scheme of recruitment to polysomes and translational activation. However, selecting and regulating individual mRNAs for the translation from a competitive pool of mRNAs are little-known processes. This research shows that the maternal eukaryotic translation initiation factor 4e1b (Eif4e1b) expresses during the oocyte-to-embryo transition (OET), and maternal deletion of Eif4e1b leads to multiple defects concerning oogenesis and embryonic developmental competence during OET. The linear amplification of complementary deoxyribonucleic acid (cDNA) ends, and sequencing (LACE-seq) is used to identify the distinct subset of mRNA and its CG-rich binding sites within the 5' untranslated region (UTR) targeted by eIF4E1B. The proteomics analyses indicate that eIF4E1B-specific bound genes show stronger downregulation at the protein level, which further verify a group of proteins that plays a crucial role in oocyte maturation and embryonic developmental competence is insufficiently synthesized in Eif4e1b-cKO oocytes during OET. Moreover, the biochemical results in vitro are combined to further confirm the maternal-specific translation activation model assembled by eIF4E1B and 3'UTR-associated mRNA binding proteins. The findings demonstrate the indispensability of eIF4E1B for selective translation activation in mammalian oocytes and provide a potential network regulated by eIF4E1B in OET., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Poly(A)-tail-mediated post-transcriptional regulation of maternal mRNAs is vital in the oocyte-to-embryo transition (OET). Nothing is known about poly(A) tail dynamics during the human OET. Here, we show that poly(A) tail length and internal non-A residues are highly dynamic during the human OET, using poly(A)-inclusive RNA isoform sequencing (PAIso-seq). Unexpectedly, maternal mRNAs undergo global remodeling: after deadenylation or partial degradation into 3'-UTRs, they are re-polyadenylated to produce polyadenylated degradation intermediates, coinciding with massive incorporation of non-A residues, particularly internal long consecutive U residues, into the newly synthesized poly(A) tails. Moreover, TUT4 and TUT7 contribute to the incorporation of these U residues, BTG4-mediated deadenylation produces substrates for maternal mRNA re-polyadenylation, and TENT4A and TENT4B incorporate internal G residues. The maternal mRNA remodeling is further confirmed using PAIso-seq2. Importantly, maternal mRNA remodeling is essential for the first cleavage of human embryos. Together, these findings broaden our understanding of the post-transcriptional regulation of maternal mRNAs during the human OET., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m 6 A, but repressed by m 5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m 6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance., (© 2022 The Authors.)

Posttranscriptional modification plays an important role in key embryonic processes. Adenosine-to-inosine RNA editing, a common example of such modifications, is widespread in human adult tissues and has various functional impacts and clinical consequences. However, whether it persists in a consistent pattern in most human embryos, and whether it supports embryonic development, are poorly understood. To address this problem, we compiled the largest human embryonic editome from 2,071 transcriptomes and identified thousands of recurrent embryonic edits (>=50% chances of occurring in a given stage) for each early developmental stage. We found that these recurrent edits prefer exons consistently across stages, tend to target genes related to DNA replication, and undergo organized loss in abnormal embryos and embryos from elder mothers. In particular, these recurrent edits are likely to enhance maternal mRNA clearance, a possible mechanism of which could be introducing more microRNA binding sites to the 3'-untranslated regions of clearance targets. This study suggests a potentially important, if not indispensable, role of RNA editing in key human embryonic processes such as maternal mRNA clearance; the identified editome can aid further investigations., (© 2022. The Author(s).)

Many maternal mRNAs are translationally repressed during oocyte development and spatio-temporally activated during early embryogenesis, which is crucial for oocyte and early embryo development. By analyzing maternal mutants of nanog (Mnanog) in zebrafish, we demonstrated that Nanog tightly controls translation of maternal mRNA during oogenesis via transcriptional repression of eukaryotic translation elongation factor 1 alpha 1, like 2 (eef1a1l2). Loss of maternal Nanog led to defects of egg maturation, increased endoplasmic reticulum stress, and an activated unfold protein response, which was caused by elevated translational activity. We further demonstrated that Nanog, as a transcriptional repressor, represses the transcription of eefl1a1l2 by directly binding to the eef1a1l2 promoter in oocytes. More importantly, depletion of eef1a1l2 in nanog mutant females effectively rescued the elevated translational activity in oocytes, oogenesis defects and embryonic defects of Mnanog embryos. Thus, our study demonstrates that maternal Nanog regulates oogenesis and early embryogenesis through translational control of maternal mRNA via a mechanism whereby Nanog acts as a transcriptional repressor to suppress transcription of eef1a1l2., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: RNA modification-induced ovarian dysgenesis appears to be necessary for ovary development. However, how m 5 C (5-methylcytosine)-coordinating modificatory transcripts are dynamically regulated during oogenesis, and ovarian development is unknown. The purpose of this study was to determine whether NOP2/Sun RNA methyltransferase 5 (Nsun5) deletion leads to suppression of ovarian function and arrest of embryonic development. The regulation of mRNA decay and stability by m 5 C modification is essential at multiple stages during the maternal-to-zygotic (MZT) transition., Methods: Mouse ovaries and oocytes with Nsun5 KO and the KGN cell line were subjected to m 5 C identification, alternative splicing analysis and protein expression. BS-m 5 C-seq, real-time polymerase chain reaction, Western blot, immunofluorescence and actinomycin D treatment assays were used. In particular, BS-m 5 C-seq revealed a dynamic pattern of m 5 C sites and genes in the ovaries between Nsun5 KO and WT mice at the 2-month and 6-month stages. Diverse bioinformatic tools were employed to identify target genes for Nsun5., Results: Here, a maternal mRNA stability study showed that deletion of the m 5 C methyltransferase Nsun5 obstructs follicular development and ovarian function, which leads directly to inhibition of embryogenesis and embryo development. Dynamic analysis of m 5 C revealed that the level of m 5 C decreased in a time-dependent manner after Nsun5 knockout. Regarding the molecular mechanism, we found that Nsun5 deficiency caused a m 5 C decline in the exon and 3'UTR regions that influenced the translation efficiency of Mitotic arrest deficient 2 like 2 (MAD2L2) and Growth differentiation factor 9 (GDF9) in the ovary. Mechanistic investigation of alternative splicing indicated that Nsun5 KO triggers aberrant events in the exon region of Brd8., Conclusions: Nsun5 loss arrests follicular genesis and development in ovarian aging, indicating that Nsun5/m 5 C-regulated maternal mRNA stabilization is essential for MZT transition., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Full-grown oocytes are transcriptionally silent and must stably maintain the messenger RNAs (mRNAs) needed for oocyte meiotic maturation and early embryonic development. However, where and how mammalian oocytes store maternal mRNAs is unclear. Here, we report that mammalian oocytes accumulate mRNAs in a mitochondria-associated ribonucleoprotein domain (MARDO). MARDO assembly around mitochondria was promoted by the RNA-binding protein ZAR1 and directed by an increase in mitochondrial membrane potential during oocyte growth. MARDO foci coalesced into hydrogel-like matrices that clustered mitochondria. Maternal mRNAs stored in the MARDO were translationally repressed. Loss of ZAR1 disrupted the MARDO, dispersed mitochondria, and caused a premature loss of MARDO-localized mRNAs. Thus, a mitochondria-associated membraneless compartment controls mitochondrial distribution and regulates maternal mRNA storage, translation, and decay to ensure fertility in mammals.

How the germ line achieves a clean transition from maternal to zygotic gene expression control is a fundamental problem in sexually reproducing organisms. Whereas several mechanisms terminate the maternal program in the soma, this combined molecular reset and handover are poorly understood for primordial germ cells (PGCs). Here, we show that GRIF-1, a TRIM32-related and presumed E3 ubiquitin ligase in Caenorhabditis elegans , eliminates the maternal cytoplasmic poly(A) polymerase (cytoPAP) complex by targeting the germline-specific intrinsically disordered region of its enzymatic subunit, GLD-2, for proteasome-mediated degradation. Interference with cytoPAP turnover in PGCs causes frequent transgenerational sterility and, eventually, germline mortality. Hence, positively acting maternal RNA regulators are cleared via the proteasome system to avoid likely interference between maternal and zygotic gene expression programs to maintain transgenerational fertility and acquire germline immortality. This strategy is likely used in all animals that preform their immortal germ line via maternally inherited germplasm determinants.

Postmeiotic spermatids use a unique strategy to coordinate gene expression with morphological transformation, in which transcription and translation take place at separate developmental stages, but how mRNAs stored as translationally inert messenger ribonucleoproteins in developing spermatids become activated remains largely unknown. Here, we report that the RNA binding protein FXR1, a member of the fragile X-related (FXR) family, is highly expressed in late spermatids and undergoes liquid-liquid phase separation (LLPS) to merge messenger ribonucleoprotein granules with the translation machinery to convert stored mRNAs into a translationally activated state. Germline-specific Fxr1 ablation in mice impaired the translation of target mRNAs and caused defective spermatid development and male infertility, and a phase separation-deficient FXR1 L351P mutation in Fxr1 knock-in mice produced the same developmental defect. These findings uncover a mechanism for translational reprogramming with LLPS as a key driver in spermiogenesis.

A decrease in oocyte developmental potential is a major obstacle for successful pregnancy in women of advanced age. However, the age-related epigenetic modifications associated with dynamic transcriptome changes, particularly meiotic maturation-coupled mRNA clearance, have not been adequately characterized in human oocytes. This study demonstrates a decreased storage of transcripts encoding key factors regulating the maternal mRNA degradome in fully grown oocytes of women of advanced age. A similar defect in meiotic maturation-triggered mRNA clearance is also detected in aged mouse oocytes. Mechanistically, the epigenetic and cytoplasmic aspects of oocyte maturation are synchronized in both the normal development and aging processes. The level of histone H3K4 trimethylation (H3K4me3) is high in fully grown mouse and human oocytes derived from young females but decreased during aging due to the decreased expression of epigenetic factors responsible for H3K4me3 accumulation. Oocyte-specific knockout of the gene encoding CxxC-finger protein 1 (CXXC1), a DNA-binding subunit of SETD1 methyltransferase, causes ooplasm changes associated with accelerated aging and impaired maternal mRNA translation and degradation. These results suggest that a network of CXXC1-maintained H3K4me3, in association with mRNA decay competence, sets a timer for oocyte deterioration and plays a role in oocyte aging in both mouse and human oocytes., (© 2022. The Author(s).)

m 5 C is one of the longest-known RNA modifications, however, its developmental dynamics, functions, and evolution in mRNAs remain largely unknown. Here, we generate quantitative mRNA m 5 C maps at different stages of development in 6 vertebrate and invertebrate species and find convergent and unexpected massive methylation of maternal mRNAs mediated by NSUN2 and NSUN6. Using Drosophila as a model, we reveal that embryos lacking maternal mRNA m 5 C undergo cell cycle delays and fail to timely initiate maternal-to-zygotic transition, implying the functional importance of maternal mRNA m 5 C. From invertebrates to the lineage leading to humans, two waves of m 5 C regulatory innovations are observed: higher animals gain cis-directed NSUN2-mediated m 5 C sites at the 5' end of the mRNAs, accompanied by the emergence of more structured 5'UTR regions; humans gain thousands of trans-directed NSUN6-mediated m 5 C sites enriched in genes regulating the mitotic cell cycle. Collectively, our studies highlight the existence and regulatory innovations of a mechanism of early embryonic development and provide key resources for elucidating the role of mRNA m 5 C in biology and disease., (© 2022. The Author(s).)

Fertilization triggers physiological degradation of maternal-mRNAs, which are then replaced by embryonic transcripts. Ample evidence suggests that Argonaut 2 (AGO2) is a possible post-fertilization regulator of maternal-mRNAs degradation; but its role in degradation of maternal-mRNAs during oocyte maturation remains obscure. Fyn, a member of the Src family kinases (SFKs), and an essential factor in oocyte maturation, was reported to inhibit AGO2 activity in oligodendrocytes. Our aim was to examine the role of Fyn and AGO2 in degradation of maternal-mRNAs during oocyte maturation by either suppressing their activity with SU6656 - an SFKs inhibitor; or by microinjecting DN-Fyn RNA for suppression of Fyn and BCl-137 for suppression of AGO2. Batches of fifteen mouse oocytes or embryos were analyzed by qPCR to measure the expression level of nine maternal-mRNAs that were selected for their known role in oocyte growth, maturation and early embryogenesis. We found that Fyn/SFKs are involved in maintaining the stability of at least four pre-transcribed mRNAs in oocytes at the germinal vesicle (GV) stage, whereas AGO2 had no role at this stage. During in-vivo oocyte maturation, eight maternal-mRNAs were significantly degraded. Inhibition of AGO2 prevented the degreadation of at least five maternal-mRNAs, whereas inhibition of Fyn/SFK prevented degradation of at least five Fyn maternal-mRNAs and two SFKs maternal-mRNAs; pointing at their role in promoting the physiological degradation which occurs during in-vivo oocyte maturation. Our findings imply the involvement of Fyn/SFKs in stabilization of maternal-mRNA at the GV stage and the involvement of Fyn, SFKs and AGO2 in degradation of maternal mRNAs during oocyte maturation.

Translational regulation plays an important role in gene expression and function. Although the transcriptional dynamics of mouse preimplantation embryos have been well characterized, the global mRNA translation landscape and the master regulators of zygotic genome activation (ZGA) remain unknown. Here, by developing and applying a low-input ribosome profiling (LiRibo-seq) technique, we profiled the mRNA translation landscape in mouse preimplantation embryos and revealed the translational dynamics during mouse preimplantation development. We identified a marked translational transition from MII oocytes to zygotes and demonstrated that active translation of maternal mRNAs is essential for maternal-to-zygotic transition (MZT). We further showed that two maternal factors, Smarcd2 and Cyclin T2, whose translation is activated in zygotes, are required for chromatin reprogramming and ZGA, respectively. Our study thus not only filled in a knowledge gap on translational regulation during mammalian preimplantation development but also revealed insights into the critical function of maternal mRNA translation in MZT.

Mammalian oocytes are transcriptionally quiescent, and meiosis and early embryonic divisions rely on translation of stored maternal mRNAs. Activation of these mRNAs is mediated by polyadenylation. Cytoplasmic polyadenylation binding element 1 (CPEB1) regulates mRNA polyadenylation. One message is aurora kinase C (Aurkc), encoding a protein that regulates chromosome segregation. We previously demonstrated that AURKC levels are upregulated in oocytes lacking aurora kinase B (AURKB), and this upregulation caused increased aneuploidy rates, a role we investigate here. Using genetic and pharmacologic approaches, we found that AURKB negatively regulates CPEB1-dependent translation of many messages. To determine why translation is increased, we evaluated aurora kinase A (AURKA), a kinase that activates CPEB1 in other organisms. We find that AURKA activity is increased in Aurkb knockout mouse oocytes and demonstrate that this increase drives the excess translation. Importantly, removal of one copy of Aurka from the Aurkb knockout strain background reduces aneuploidy rates. This study demonstrates that AURKA is required for CPEB1-dependent translation, and it describes a new AURKB requirement to maintain translation levels through AURKA, a function crucial to generating euploid eggs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The goal of preimplantation development is to establish the fates of the embryonic and extra-embryonic cells. However, when and how cell fates are determined during early mammalian embryonic development remains unclear. We report that the high mobility group (HMG) protein family member HMGA1 was distributed differentially in mouse two-cell blastomeres. Knockdown of Hmga1 expression in one of the two cells reduced the number of cells contributing to the inner cell mass (ICM), suggesting that differential distribution of HMGA1 in the blastomeres in two-cell mouse embryos affected the selection of embryonic cell lineages. Mechanistically, HMGA1 promotes the expression of the ICM-specific gene Sox2. The results of this study show that mouse embryos demonstrate heterogeneity as early as the two-cell stage, and that these differences are related to cell-fate differentiation in early mouse embryos., (© 2021 Federation of American Societies for Experimental Biology.)

Ribonucleoprotein condensates can exhibit diverse physical states in vitro and in vivo. Despite considerable progress, the relevance of condensate physical states for in vivo biological function remains limited. Here, we investigated the physical properties of processing bodies (P bodies) and their impact on mRNA storage in mature Drosophila oocytes. We show that the conserved DEAD-box RNA helicase Me31B forms viscous P body condensates, which adopt an arrested physical state. We demonstrate that structurally distinct proteins and protein-protein interactions, together with RNA, regulate the physical properties of P bodies. Using live imaging and in situ hybridization, we show that the arrested state and integrity of P bodies support the storage of bicoid (bcd) mRNA and that egg activation modulates P body properties, leading to the release of bcd for translation in the early embryo. Together, this work provides an example of how physical states of condensates regulate cellular function in development., Competing Interests: Declarations of interests S.A. is an advisor on the scientific advisory board of Dew point Therapeutics. A.S.H. is a scientific consultant with Dewpoint Therapeutics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Control of mRNA translation is a key mechanism by which the differentiated oocyte transitions to a totipotent embryo. In Drosophila , the PNG kinase complex regulates maternal mRNA translation at the oocyte-to-embryo transition. We previously showed that the GNU activating subunit is crucial in regulating PNG and timing its activity to the window between egg activation and early embryogenesis (Hara et al., 2017). In this study, we find associations between GNU and proteins of RNP granules and demonstrate that GNU localizes to cytoplasmic RNP granules in the mature oocyte, identifying GNU as a new component of a subset of RNP granules. Furthermore, we define roles for the domains of GNU. Interactions between GNU and the granule component BIC-C reveal potential conserved functions for translational regulation in metazoan development. We propose that by binding to BIC-C, upon egg activation GNU brings PNG to its initial targets, translational repressors in RNP granules., Competing Interests: EA, MH, TO No competing interests declared, (© 2021, Avilés-Pagán et al.)

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3' untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3' UTR reporter as an example.

Mammalian oocyte maturation is driven by strictly regulated polyadenylation and translational activation of maternal mRNA stored in the cytoplasm. However, the poly(A) polymerase (PAP) that directly mediates cytoplasmic polyadenylation in mammalian oocytes has not been determined. In this study, we identified PAPα as the elusive enzyme that catalyzes cytoplasmic mRNA polyadenylation implicated in mouse oocyte maturation. PAPα was mainly localized in the germinal vesicle (GV) of fully grown oocytes but was distributed to the ooplasm after GV breakdown. Inhibition of PAPα activity impaired cytoplasmic polyadenylation and translation of maternal transcripts, thus blocking meiotic cell cycle progression. Once an oocyte resumes meiosis, activated CDK1 and ERK1/2 cooperatively mediate the phosphorylation of three serine residues of PAPα, 537, 545 and 558, thereby leading to increased activity. This mechanism is responsible for translational activation of transcripts lacking cytoplasmic polyadenylation elements in their 3'-untranslated region (3'-UTR). In turn, activated PAPα stimulated polyadenylation and translation of the mRNA encoding its own (Papola) through a positive feedback circuit. ERK1/2 promoted Papola mRNA translation in a 3'-UTR polyadenylation signal-dependent manner. Through these mechanisms, PAPα activity and levels were significantly amplified, improving the levels of global mRNA polyadenylation and translation, thus, benefiting meiotic cell cycle progression., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During the maternal-to-zygotic transition (MZT), multiple mechanisms precisely control massive decay of maternal mRNAs. N 6 -methyladenosine (m 6 A) is known to regulate mRNA decay, yet how this modification promotes maternal transcript degradation remains unclear. Here, we find that m 6 A promotes maternal mRNA deadenylation. Yet, genetic loss of m 6 A readers Ythdf2 and Ythdf3 did not impact global maternal mRNA clearance, zygotic genome activation, or the onset of gastrulation, challenging the view that Ythdf2 alone is critical to developmental timing. We reveal that Ythdf proteins function redundantly during zebrafish oogenesis and development, as double Ythdf2 and Ythdf3 deletion prevented female gonad formation and triple Ythdf mutants were lethal. Finally, we show that the microRNA miR-430 functions additively with methylation to promote degradation of common transcript targets. Together these findings reveal that m 6 A facilitates maternal mRNA deadenylation and that multiple pathways and readers act in concert to mediate these effects of methylation on RNA stability., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3'-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.

During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Bansal et al.)

Early embryogenesis relies on maternally inherited mRNAs. Although the mechanism of maternal mRNA degradation during maternal-to-zygotic transition (MZT) has been extensively studied in vertebrates, how the embryos maintain maternal mRNA stability remains unclear. Here, we identify Igf2bp3 as an important regulator of maternal mRNA stability in zebrafish. Depletion of maternal igf2bp3 destabilizes maternal mRNAs prior to MZT and leads to severe developmental defects, including abnormal cytoskeleton organization and cell division. However, the process of oogenesis and the expression levels of maternal mRNAs in unfertilized eggs are normal in maternal igf2bp3 mutants. Gene ontology analysis revealed that these functions are largely mediated by Igf2bp3-bound mRNAs. Indeed, Igf2bp3 depletion destabilizes while its overexpression enhances its targeting maternal mRNAs. Interestingly, igf2bp3 overexpression in wild-type embryos also causes a developmental delay. Altogether, these findings highlight an important function of Igf2bp3 in controlling early zebrafish embryogenesis by binding and regulating the stability of maternal mRNAs.

In animals, the early embryo is mostly transcriptionally silent and development is fueled by maternally supplied mRNAs and proteins. These maternal products are important not only for survival, but also to gear up the zygote's genome for activation. Over the last three decades, research with different model organisms and experimental approaches has identified molecular factors and proposed mechanisms for how the embryo transitions from being transcriptionally silent to transcriptionally competent. In this chapter, we discuss the molecular players that shape the molecular landscape of ZGA and provide insights into their mode of action in activating the transcription program in the developing embryo., (© 2020 Elsevier Inc. All rights reserved.)

The life cycle of many organisms includes a quiescent stage, such as bacterial or fungal spores, insect larvae, or plant seeds. Common to these stages is their low water content and high survivability during harsh conditions. Upon rehydration, organisms need to reactivate metabolism and protein synthesis. Plant seeds contain many mRNAs that are transcribed during seed development. Translation of these mRNAs occurs during early seed germination, even before the requirement of transcription. Therefore, stored mRNAs are postulated to be important for germination. How these mRNAs are stored and protected during long-term storage is unknown. The aim of this study was to investigate how mRNAs are stored in dry seeds and whether they are indeed translated during seed germination. We investigated seed polysome profiles and the mRNAs and protein complexes that are associated with these ribosomes in seeds of the model organism Arabidopsis ( Arabidopsis thaliana ). We showed that most stored mRNAs are associated with monosomes in dry seeds; therefore, we focus on monosomes in this study. Seed ribosome complexes are associated with mRNA-binding proteins, stress granule, and P-body proteins, which suggests regulated packing of seed mRNAs. Interestingly, ∼17% of the mRNAs that are specifically associated with monosomes are translationally up-regulated during seed germination. These mRNAs are transcribed during seed maturation, suggesting a role for this developmental stage in determining the translational fate of mRNAs during early germination., (© 2020 The author(s). All Rights Reserved.)

Maternal genes play an important role in the early embryonic development of the silkworm. Early embryonic development without new transcription depends on maternal components stored in the egg during oocyte maturation. The maternal-to-zygotic transition (MZT) is a tightly regulated process that includes maternal mRNAs elimination and zygotic transcription initiation. This process has been extensively studied within model species. Each model organism has a unique pattern of maternal transcriptional clearance classes in MZT. In this study, we identified 66 maternal genes through bioinformatics analysis and expression analysis in the eggs of silkworm virgin moths ( Bombyx mori ). All 66 maternal genes were expressed in vitellogenesis in day eight female pupae. During MZT, the degradation of maternal gene mRNAs could be divided into three clusters. We found that eight maternal genes of cluster 1 remained stable from 0 to 3.0 h, 17 maternal genes of cluster 2 were significantly decayed from 0.5 to 1.0 h and 41 maternal genes of cluster 3 were significantly decayed after 1.5 h. Therefore, the initial time-point of degradation of cluster 2 was earlier than that of cluster 3. The maternal gene mRNAs decay of clusters 2 and 3 is first initiated by maternal degradation activity. Our study expands upon the identification of silkworm maternal genes and provides a perspective for further research of the embryo development in Bombyx mori .

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m 5 C) modification in zebrafish early embryos, we found that m 5 C-modified maternal mRNAs display higher stability than non-m 5 C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m 5 C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m 5 C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m 5 C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m 5 C mRNA modification in early development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In mammals, maternal-to-zygotic transition (MZT), or oocyte-to-embryo transition, begins with oocyte meiotic resumption due to the sequential translational activation and destabilization of dormant maternal transcripts stored in the ooplasm. It then continues with the elimination of maternal transcripts during oocyte maturation and fertilization and ends with the full transcriptional activation of the zygotic genome during embryonic development. A hallmark of MZT in mammals is its reliance on translation and the utilization of stored RNAs and proteins, rather than de novo transcription of genes, to sustain meiotic maturation and early development. Impaired maternal mRNA clearance at the onset of MZT prevents zygotic genome activation and causes early arrest of developing embryos. In this review, we discuss recent advances in our knowledge of the mechanisms whereby mRNA translation and degradation are controlled by cytoplasmic polyadenylation and deadenylation which set up the competence of maturing oocyte to accomplish MZT. The emphasis of this review is on the mouse as a model organism for mammals and BTG4 as a licensing factor of MZT under the translational control of the MAPK cascade., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Ionizing radiation is administered to damage nuclear genome in fish eggs during induced androgenesis. In this study, we examined whether 350 Gy of X-ray applied to damage chromosomes in the rainbow trout eggs affects maternal RNA. Shortly after irradiation, we did not find any symptoms of RNA degradation in the treated eggs. Significant (p < 0.01) differences between non-irradiated and irradiated eggs concerned only a few transcripts including increased expression of immediate early response 2 (IER2) and early growth response 1 (EGR1) genes observed in the irradiated eggs. Both genes belong to the group of "immediate early genes" that respond quickly to the diverse extracellular stimuli. Elevated expression of these genes was accompanied by decreased level of ssa-miR-10b-5p and ssa-miR-21b-5p (p < 0.05), for which IER2 and EGR1 are target genes. The level of RNA in the fertilized irradiated eggs was highly significantly lower than in the non-irradiated eggs (p < 0.001) and in the unfertilized irradiated eggs (p < 0.0001). However, transcriptome profiles of fertilized non-irradiated eggs and fertilized irradiated eggs did not differ significantly. Thus, we assume that reduced abundance of mRNA in the fertilized irradiated eggs was associated with post-translational degradation and clearance of the maternal transcripts rather than from the irradiation of eggs.

Transcription ceases upon stimulation of oocyte maturation and gene expression during oocyte maturation, fertilization, and early cleavage relies on translational activation of maternally derived mRNAs. Two key mechanisms that mediate translation of mRNAs in oocytes have been described in detail: cytoplasmic polyadenylation-dependent and -independent. Both of these mechanisms utilize specific protein complexes that interact with cis-acting sequences located on 3'-untranslated region (3'-UTR), and both involve embryonic poly(A) binding protein (EPAB), the predominant poly(A) binding protein during early development. While mechanistic details of these pathways have primarily been elucidated using the Xenopus model, their roles are conserved in mammals and targeted disruption of key regulators in mouse results in female infertility. Here, we provide a detailed account of the molecular mechanisms involved in translational activation during oocyte and early embryo development, and the role of EPAB in this process., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)