Genome-wide analysis reveals a switch in the translational program upon oocyte meiotic resumption

Abstract

Cell development relies on elaborate changes in gene expression in order to transition 38 through different phenotypic and functional stages that ultimately lead to terminal differentiation. 39Changes in gene expression are achieved through transcriptional and post-transcriptional 40 regulations. Although transcriptional regulation is understood in considerable detail 1, 2 , much less 41 is known about the molecular machinery involved in translation regulation. 42Large oligomeric complexes involving proteins and non-coding RNAs are assembled on 43 the mRNA 3 to regulate its interaction with ribosomes, its translation rate, and its stability 4, 5 . In 44 somatic cells, numerous observations indicate that translation is intimately coupled with 45 degradation of mRNAs 5, 6 . Proteins recruited on the mRNA interact with elements located 46 throughout the length of the transcript 3, 7 . However, complexes nucleated around the 5' and 3' 47 polyadenylation element-binding protein (CPEB) is considered a master regulator of 63 polyadenylation and translation 18,19 . Much less is known about the role of CPEB in mammalian 64 oocytes. Here, we have used a genome-wide approach to investigate the role of this RNA-binding 65 protein (RBP) during the transition from quiescence to re-entry into the meiosis. Through a 66 detailed time course, we have investigated the temporal association between maternal mRNA 67 translation and the different steps involved in oocyte re-entry into and progression through the 68 meiosis. Using a RiboTag/RNA-Seq strategy, we describe a genome-wide switch in the 69 translation program of maternal mRNAs, and define new, critical functions of CPEB in the control 70 of this switch. 71 4 Results 72 Re-entry into meiosis coincides with rapid translational changes of stable mRNAs 73We have used a RiboTag/RNA-Seq strategy to characterize mRNA translation in oocytes 74 arrested at prophase I (GV) and in those undergoing meiotic maturation. (Fig. 1a). This strategy 75 has been previously validated 20, 21 and additional quality controls are reported (Supplementary 76 Fig. 1a-d). Upon meiotic resumption, there were both progressive increases and decreases in 77 ribosome loading of maternal mRNAs (Fig. 1b). By late metaphase I (MI), mRNAs were either 78 constitutively translated (n = 5580, CONSTITUTIVE), translationally repressed (n = 1092, 79 DOWN), or translationally activated (n = 871, UP) (FDR ≤ 0.05 and -1 ≥ log2(FC) (LFC) ≥ 1, 80 Supplementary Fig. 1e). Total mRNA levels remained stable up to MI and significant 81 destabilization was only detectable for 3% of maternal mRNAs at late MI ( Fig. 1b and 82 Supplementary Fig. 1f). Comparison of changes in total mRNA levels (transcriptome) to changes 83 in ribosome-associated mRNA levels (translatome) confirms this late MI destabilization (Fig. 1c), 84 which became prominent later during MII arrest, with a subset of mRNAs remaining stable even 85 if their translation was repressed. Changes in translation that initiate during MI were extended into 86 and amplified ...

The RNA-binding protein DAZL functions as repressor and activator of mRNA translation during oocyte maturation

“…To obtain further insight into the mechanism of DAZL repression of translation, we used a novel strategy where the mutagenesis of RBP binding elements and translation of oligoadenylated or poly-adenylated reporters are combined. If a reporter is repressed in the GV state, the translation rate of an oligo-adenylated probe remains constant 32 . If repression is relieved, as it happens when an RBP binding site is mutated, an increase in translation of oligo-adenylated probes becomes evident with time 32 .…”

“…If a reporter is repressed in the GV state, the translation rate of an oligo-adenylated probe remains constant 32 . If repression is relieved, as it happens when an RBP binding site is mutated, an increase in translation of oligo-adenylated probes becomes evident with time 32 . Moreover, forcing polyadenylation of a repressed reporter causes a transient increase in translation that is reversed over time because the reporter becomes de-adenylated 32 .…”

“…If repression is relieved, as it happens when an RBP binding site is mutated, an increase in translation of oligo-adenylated probes becomes evident with time 32 . Moreover, forcing polyadenylation of a repressed reporter causes a transient increase in translation that is reversed over time because the reporter becomes de-adenylated 32 . Using this experimental paradigm, we find that combined mutagenesis of the DAZL and CPE element in Oosp1-short NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15209-9 ARTICLE 3′UTR causes maximum increase in translation of the oligoadenylated reporter (Fig.…”

Age‐related differences in the translational landscape of mammalian oocytes

“…In order to identify polysome-bound mRNAs in mouse oocytes, we previously developed a method that enables isolation of very small amounts of polyribosomal bound RNA for a subsequent genome-wide analysis (Ganesh et al, 2020;Masek et al, 2020). To date, there have been very few experiments applying polysome profiling techniques to oocytes (Chen et al, 2011;Luong, Daldello, Rajkovic, Yang, & Conti, 2020;Potireddy et al, 2006;Scantland et al, 2011), and to our knowledge, we are the first to use it to interrogate the whole translatome in oocytes of different maternal ages.…”

Tdrd3 regulates the progression of meiosis II through translational control ofEmi2mRNA in mouse oocytes

“…After the resumption of meiosis, hundreds of mRNAs begin to be translated in meiosis I and more than one thousand mRNAs begin to be translated in meiosis II (Chen et al, 2011;Luong et al, 2020). cyclin B1 mRNA is one of the mRNAs translated in early meiosis I (Haccard and Jessus, 2006;Han et al, 2017;Kotani et al, 2013;Nakahata et al, 2003;Yang et al, 2017).…”