Ultrasensitive Ribo-seq reveals translational landscapes during mammalian oocyte-to-embryo transition and pre-implantation development
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ABSTRACT: In mammals, translational control plays critical roles during oocyte-to-embryo transition (OET) when transcription ceases. However, the underlying regulatory mechanisms remain challenging to study. Here, using low-input Ribo-seq (Ribo-lite), we investigated translational landscapes during OET using 30-150 mouse oocytes or embryos per stage. Ribo-lite can also accommodate single oocytes. Combining PAIso-seq to interrogate poly(A) tail lengths, we found a global switch of translatome that closely parallels changes of poly(A) tails upon meiotic resumption. Translation activation correlates with polyadenylation and is supported by polyadenylation signal proximal cytoplasmic polyadenylation elements (papCPEs) in 3' untranslated regions. By contrast, translation repression parallels global de-adenylation. The latter includes transcripts containing no CPEs or non-papCPEs, which encode many transcription regulators that are preferentially re-activated before zygotic genome activation. CCR4-NOT, the major de-adenylation complex, and its key adaptor protein BTG4 regulate translation downregulation often independent of RNA decay. BTG4 is not essential for global de-adenylation but is required for selective gene de-adenylation and production of very short-tailed transcripts. In sum, our data reveal intimate interplays among translation, RNA stability and poly(A) tail length regulation underlying mammalian OET. Note: RNA-seq data for PN5 zygotes, early two-cell, late two-cell, four-cell and eight-cell stages and ICM were obtained from our previous publication (Zhang et al., Nature, 2016: Allelic reprogramming of the histone modification H3K4me3 in early mammalian development, GSE71434).
Project description:Translational regulation plays a critical role during oocyte-to-embryo transition including zygotic genome activation. However, the translatome during OET and molecular mechanisms underlying human ZGA remain largely uncharted. Here, we integrated ultra-low-input Ribo-seq with RNA-seq (R2-lite) to jointly profile the translatome and transcriptome from the same samples across 8 stages in human oocytes and early embryos. These data not only unveil conservation and divergence of the translation landscapes between human and mouse OET, but also identify critical regulators of human ZGA.
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:The Poly(A)-Tail focused RNA-seq, or PAT-seq approach, is an affordable and efficient tool for the measure of 3’UTR dynamics. We show here that PAT-seq returns (i) digital gene expression, (ii) polyadenylation site usage within and between samples, including alternative adenylation, and (iii) the polyadenylation-state the transcriptome. PAT-seq differs from previous 3’ focused RNA-seq methods in that it strictly depends on native 3’ adenylation within total RNA samples and thus removes the need for ribosome depletion and, that the full native poly(A)-tail is included in the sequencing libraries. Limited RNase digestion combined with size selection and directional sequencing mean that deep-sequencing reads map to within ~50-100 bases of adenylation sites and run from unique sequence into adenosine homopolymers. Here, total RNA samples from budding yeast cells were analyzed to highlight the changes in gene expression and adenylation-state of the transcriptome in response to loss of the deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in a classic Crabtree-Warburg metabolic shift. Because all adenylated RNA are interrogated by the PAT-seq approach, alternative adenylation sites, long noncoding RNA and other non-coding RNA and RNA decay intermediates were also identified.
Project description:The molecular causes of deteriorating oocyte quality during aging are poorly defined. Since oocyte developmental competence relies on post-transcriptional regulations, we tested whether defective mRNA translation contributes to this decline in quality. Disruption in ribosome loading on maternal transcripts is present in old oocytes. Using a candidate approach, we detect altered translation of 3’-UTR-reporters and altered poly(A) length of the endogenous mRNAs. mRNA polyadenylation depends on the cytoplasmic polyadenylation binding protein 1 (CPEB1). Cpeb1 mRNA translation and protein levels are decreased in old oocytes. This decrease causes de-repression of Ccnb1 translation in quiescent oocytes, premature CDK1 activation, and accelerated reentry into meiosis. De-repression of Ccnb1 is corrected by Cpeb1 mRNA injection in old oocytes. Oocyte-specific Cpeb1 haploinsufficiency in young oocytes recapitulates all the translation phenotypes of old oocytes. These findings demonstrate that a dysfunction in the oocyte translation program is associated with the decline in oocyte quality during aging.
Project description:The comparison of trancriptomes was part of the study by Pasternak et al. The goal was to check if BTG4 regulates mRNA polyadenylation during mouse oocyte meiosis. To test this we compared the abundancies of the polyadenylated trancripts in control and Btg4-depleted oocytes.
Project description:We report systematical profiling of translation efficiency and mRNA stability dependent on the dynamics of poly(A)-tail length in stress conditions of human cells. In this study, we developed a new feasible method measuring poly(A)-tail length called TED-seq and applied it to investigate the change of mRNA's poly(A)-tail lengths in ER stress pharmacologically induced by thapsigargin (THAP). Combined with other global RNA analyses such as RNA-seq, Ribo-seq and PRO-seq, we observed that ER stress induced lenthening poly(A)-tail length, in particular of ER-stress-regulators, upon ER stress. More specifically, these mRNAs are translationally de-repressed and more stabilized based on increase in poly(A)-tail length. We also identified that insoluble fractions which include stress-induced RNA-granules have overall shorter length of poly(A) tail. Taken together, our data suggest that poly(A)-tail lengths are dynamically regulated and influence both translation efficiency and mRNA stability in ER stress.
Project description:The oocyte-to-embryo transition (OET) is thought to be mainly driven by post-transcriptional gene regulation. However, expression of both RNAs and proteins during the OET has not been comprehensively assayed. Furthermore, specific molecular mechanisms that regulate gene expression during OET are largely unknown. Here, we quantify and analyze, transcriptome-wide, expression of mRNAs, small RNAs and thousands of proteins in C. elegans oocytes, 1-cell, and 2-cell embryos. This represents a first comprehensive gene expression atlas during the OET in animals. We discovered a first wave of degradation in which thousands of mRNAs are turned over shortly after fertilization. Sequence analysis revealed a statistically highly significant presence of a novel polyC motif in the 3' untranslated regions (3' UTRs) of most of these degraded mRNAs. Transgenic reporter assays showed that this polyC motif is indeed required and sufficient for mRNA degradation after fertilization. We show that orthologs of human poly-C binding-protein specifically bind this motif. Together, our data suggest a mechanism in which the polyC motif and binding partners direct degradation of maternal mRNAs. Our data also indicate that endogenous siRNAs but not miRNAs promote mRNA clearance during the OET. To study the OET in C.elegans we isolated large quantities of oocytes, 1-cell embryos, 2-cell embryos and sperm. We sequenced then sequenced polyA RNA.
Project description:At the 3'-ends of genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine 1 residues of its C-terminal domain (CTD), resulting in recruitment of transcription termination factors. We show that the multisubunit cleavage and polyadenylation factor (CPF) is a Pol II CTD phosphatase and its Glc7 subunit is required for Tyr1 dephosphorylation at the poly-adenylation site and Pol II termination in vivo. ChIP-chip was performed to examine the effect of Glc7 nuclear depletion on genome-wide Pol II occupancy [using ?-Rpb3 (1Y26, cat. no. W0012, neoclone) antibody] and CTD tyrosine 1 phosphorylation levels [using ?-TyrY1P (3D12, D. Eick) antibody].
Project description:Maternal mRNA degradation is a critical event of maternal-to-zygotic transition (MZT) that determines the developmental potential of early embryos. Nuclear Poly(A)-binding proteins (PABPNs) are extensively involved in mRNA post-transcriptional regulations, but their functions in mammalian MZT has not been investigated. In this study, we identified a novel maternal-effect factor PABPN1-like (PABPN1L), rather than its ubiquitously expressed homolog PABPN1, as an RNA-binding adapter of the mammalian MZT licensing factor BTG4 which mediates maternal mRNAs clearance. Female Pabpn1l null mice produced morphologically normal oocytes but were infertile owing to early developmental arrest of the resultant embryos at the 1-to-2-cell stages without undergoing ZGA. Deletion of Pabpn1l impairs the deadenylation and degradation of a subset of BTG4-targeted maternal mRNAs during MZT. In addition to recruiting BTG4 to the mRNA 3ʹ-poly(A) tails, PABPN1L is also required for BTG4 protein accumulation in maturing oocytes by protecting BTG4 from SCF-βTrCP1 E3 ubiquitin ligase-mediated polyubiquitination and degradation. This study highlights a noncanonical cytoplasmic function of nuclear poly(A)-binding proteins mRNA turnover as well as its physiological importance during MZT.
Project description:Changes in gene expression are required to orchestrate changes in cell state during development. Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and use changes in mRNA poly-A tail length to control protein production. Recent technical advances have enabled genome-wide measurement of poly-A tail length. Poly-A tail lengthening is correlated with translational activation and poly-A tail shortening is correlated with translational repression at a global level during early development. However, it is not clear how poly-A tail changes affect mRNA translation at a global level during vertebrate oocyte maturation. We used TAIL-seq and polyribosome analysis to measure poly-A tail and translational changes during oocyte maturation in Xenopus laevis. We found that the transcriptome undergoes large-scale poly-A and translational changes during oocyte maturation and that poly-A tail length and translation are well-correlated. Interestingly, we found that poly-A tail changes precede translation changes. Additionally, we identified a family of U-rich sequence elements that are enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. Interestingly, we found that cytoplasmic polyadenylation is not sufficient to activate translation, which requires a specific density and spacing of U-rich elements. Collectively, our data shows that changes in mRNA polyadenylation are the dominant mechanism controlling protein expression during vertebrate oocyte maturation and that these changes are controlled by a group of cis-acting sequence elements. Our results provide insight into mechanisms of translational control in oocytes and identify novel proteins important for the completion of meiosis.