Project description:Eukaryotic mRNAs are subject to multiple types of tailing which critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAILseq (mRNA TAIL-seq or mTAIL-seq) with enhanced sequencing depth for mRNAs (by ~1000 fold compared to the previous version). The improved method allows us to investigate the regulation of poly(A) tail in Drosophila oocytes and embryos. We find that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAILseq data with ribosome profiling data, we find a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tail in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems. Two sets of RNA-seq on 3 developmental stages (immature oocyte, mature oocyte, and activated egg) of wild type and wispy mutant of Drosophila melanogaster.
Project description:Because maturing oocytes and early embryos lack transcription, posttranscriptional regulatory processes must control their development. To better understand this control, we profiled translational efficiencies and poly(A)-tail lengths throughout Drosophila oocyte maturation and early embryonic development. The correspondence between translational-efficiency changes and tail-length changes indicated that tail-length changes broadly reshape translational activity until gastrulation, when this coupling disappears. Relative changes were largely retained in the absence of poly(A)-tail lengthening, which indicated that selective poly(A)-tail shortening primarily specifies the changes. Many translational changes depended on PAN GU and Smaug, and both acted primarily through tail-length changes. Our results also revealed tail-lengthâindependent mechanisms of translational control that repressed translation regardless of tail-length changes during oocyte maturation, maintained translation despite tail-length shortening during oocyte maturation, and prevented detectable translation of bicoid and several other mRNAs before egg activation. In addition to these fundamental insights, our results provide valuable resources for future studies. 42 samples analyzed using RNA-seq, ribosome footprint profiling, and PAL-seq.
Project description:The oocyte-to-embryo transition (OET) occurs in the absence of new transcription and relies on post-transcriptional gene regulation, including translational control by mRNA poly(A) tail regulation, where cytoplasmic polyadenylation activates translation and deadenylation leads to translational repression and decay. However, how the transcriptome-wide landscape of mRNA poly(A) tails shapes translation across the OET in mammals remains unknown. Here, we performed long-read RNA sequencing to uncover poly(A) tail lengths and mRNA abundance transcriptome-wide in mice across five stages of development from oocyte to embryo. Integrating these data with recently published ribosome profiling data, we demonstrate that poly(A) tail length is coupled to translational efficiency across the entire OET. We uncover an extended wave of global deadenylation during fertilization, which sets up a switch in translation control between the oocyte and embryo. In the oocyte, short-tailed maternal mRNAs that resist deadenylation in the oocyte are translationally activated, whereas large groups of mRNAs deadenylated without decay in the oocyte are later readenylated to drive translation activation in the early embryo. Our findings provide an important resource and insight into the mechanisms by which cytoplasmic polyadenylation and deadenylation dynamically shape poly(A) tail length in a stage-specific manner to orchestrate development from oocyte to embryo in mammals.
Project description:In animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transits into an actively developing embryo, initiates with an increase of Ca2+ in the oocyte’s cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepare the oocyte to undertake embryogenesis. Calcineurin is a highly conserved phosphatase that is activated directly by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus, ascidians and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fzy, Greatwall (Gwl) and Endosulfine (Endos), protein translation modulators including PNG, NAT, eIF4G and eIF4B, and important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo.
Project description:In animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transits into an actively developing embryo, initiates with an increase of Ca2+ in the oocyte’s cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepare the oocyte to undertake embryogenesis. Calcineurin is a highly conserved phosphatase that is activated directly by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus, ascidians and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fzy, Greatwall (Gwl) and Endosulfine (Endos), protein translation modulators including PNG, NAT, eIF4G and eIF4B, and important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo
Project description:Because maturing oocytes and early embryos lack transcription, posttranscriptional regulatory processes must control their development. To better understand this control, we profiled translational efficiencies and poly(A)-tail lengths throughout Drosophila oocyte maturation and early embryonic development. The correspondence between translational-efficiency changes and tail-length changes indicated that tail-length changes broadly reshape translational activity until gastrulation, when this coupling disappears. Relative changes were largely retained in the absence of poly(A)-tail lengthening, which indicated that selective poly(A)-tail shortening primarily specifies the changes. Many translational changes depended on PAN GU and Smaug, and both acted primarily through tail-length changes. Our results also revealed tail-length–independent mechanisms of translational control that repressed translation regardless of tail-length changes during oocyte maturation, maintained translation despite tail-length shortening during oocyte maturation, and prevented detectable translation of bicoid and several other mRNAs before egg activation. In addition to these fundamental insights, our results provide valuable resources for future studies.