Biology subjects

Susor, A.

Publications and source records attributed to Susor, A..

10 recordsLinked to original sources

bioRxiv2024

Absence of CDK12 in oocyte leads to female infertility

Transcriptional activity and gene expression are essential for the development of a mature, meiotically competent oocyte. We have found that the absence of cyclin-dependent kinase 12 (CDK12) in oocytes leads to complete female sterility, as there are no fully developed oocytes able to accomplish meiosis I in the ovaries. Mechanistically, CDK12 in growing oocytes controls POLII activity and maintenance of the physiological maternal transcriptome, which negatively affects protein synthesis that promotes further oocyte growth. In addition, disruption of oocyte development disrupts folliculogenesis, resulting in a premature failure phenotype without terminal folliculogenesis and ovulation. In summary, we have characterized a single master regulator of the oocyte transcriptional program and gene expression that is essential for oocyte growth and female fertility. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=113 HEIGHT=200 SRC="FIGDIR/small/622910v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@179b286org.highwire.dtl.DTLVardef@b7a847org.highwire.dtl.DTLVardef@1890a35org.highwire.dtl.DTLVardef@1fd2798_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Spatiotemporal dynamics and selectivity of mRNA translation during mouse pre-implantation development

Translational regulation is pivotal during preimplantation development. However, how mRNAs are selected for temporal regulation and their dynamic utilization and fate during this period are still unknown. Using a high-resolution ribosome profiling approach, we analyzed the transcriptome, as well as monosome- and polysome-bound RNAs of mouse oocytes and embryos, defining an unprecedented extent of spatiotemporal translational landscapes during this rapid developmental phase. We observed previously unknown mechanisms of translational selectivity, i.e., stage-wise deferral of loading monosome-bound mRNAs to polysome for active translation, continuous translation of both monosome and polysome-bound mRNAs at the same developmental stage, and priming to monosomes after initial activation. We showed that a eukaryotic initiation factor Eif1ad3, which is exclusively translated in the 2-Cell embryo, is required for ribosome biogenesis post embryonic genome activation. Our study thus provides genome-wide datasets and analyses of spatiotemporal translational dynamics accompanying mammalian germ cell and embryonic development and reveals the contribution of a novel translation initiation factor to mammalian pre-implantation development.

developmental biology↗

bioRxiv2024

Pancreatic cancer cells exchange ribosomes through tunneling nanotubes

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/597772v3_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1f66716org.highwire.dtl.DTLVardef@1ea14eeorg.highwire.dtl.DTLVardef@1044da2org.highwire.dtl.DTLVardef@184d8a3_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG Key pointsO_LIpancreatic tumour cell lines and cells from patient biopsies form tunnelling nanotubes in 2D culture C_LIO_LIformation of tunnelling nanotubes is promoted by gemcitabine C_LIO_LIpolyadenylated mRNAs, ribosomal components and assembled ribosomes are present in tunnelling nanotubes C_LIO_LIribosomes and their components are transferred via nanotubes to acceptor cells C_LIO_LIsilencing of ribosomal proteins S6 and L24 reduces the number of assembled ribosomes C_LIO_LIglobal protein synthesis and number of ribosomes in pancreatic cancer cells with silenced ribosomal proteins increases when co-cultured with translationally unimpaired cells C_LI BackgroundPancreatic ductal adenocarcinoma (PDAC) is considered as one of the deadliest types of cancer. Tunnelling nanotubes (TNTs) are thin, membranous, intercellular communication structures observed in normal and cancer cells, where they mediate the exchange of intracellular material and promote cell fitness, cancer spread and treatment resistance. ResultsPDAC cells increase the formation of TNTs upon exposure to gemcitabine. In the PANC-1 cell line and in tumour explants from patients, we observe polyadenylated mRNA, 5.8S rRNA, ribosomal proteins and assembled 80S ribosomes within the TNTs. Using HaloTag-labelled small ribosomal subunit component RPS9 we demonstrate the transport of ribosomes via TNTs into acceptor cells. Downregulation of ribosomal proteins S6 and L24 decreases the number of assembled ribosomes and the global protein translation in PDAC cells, while a co-culture with translationally unimpaired cells partially restores protein synthesis in cells with impaired protein translation. ConclusionsPDAC cells can exchange components of the protein translation machinery and mRNA. The intercellular transfer of these components causes a partial restoration of protein translation in cells with impaired protein synthesis, which may contribute to the resilience of pancreatic cancer cells, highlighting the potential of targeting TNT dynamics as a therapeutic approach for PDAC.

cancer biology↗

bioRxiv2023

Spatial positioning of preimplantation mouse embryo blastomeres is regulated by mTORC1 and 7mG-cap dependent translation at the 8- to 16-cell transition.

Preimplantation stages of mouse embryo development involve temporal and spatial specification and segregation of three late blastocyst cell lineages; trophectoderm (TE), primitive endoderm (PrE) and epiblast (EPI). Spatial separation of the outer TE lineage from the two inner cell mass (ICM) lineages (PrE and EPI) starts with the 8- to 16-cell transition and concludes following transit through the 16- to 32-cell stages. This results in an early blastocyst ICM derived from descendants of primary founding inner cells and a secondarily contributed population, of which subsequent relative EPI versus PrE potencies are subject to debate. Here, we report generation of primary but not the secondary ICM populations is highly dependent on temporally discreet activation of the mammalian target of Rapamycin (mTOR - specifically mTORC1) during M-phase entry at the 8-cell stage. This role is mediated via regulation of the 7-methylguanosine-(7mG) cap binding initiation complex (EIF4F), linked to translation of a subset of key mRNAs containing 5 UTR terminal oligopyrimidine (TOP-) or TOP-like sequence motifs; as knockdown of identified TOP-like motif containing transcripts also impairs generation of 16-cell stage primary ICM founders. However, mTOR inhibition induced ICM cell number deficits at the early blastocyst stage can be compensated by the late blastocyst stage, in the absence of inhibition. This compensation is likely initiated at the 32-cell stage when supernumerary outer cells in mTOR-inhibited embryos exhibit molecular characteristics of inner cells. Collectively, the data identify a novel mechanism specifically governing initial spatial segregation of blastomeres in the mouse embryo, that is distinct from those directing subsequent inner cell formation and contributes to germane segregation of late blastocyst lineages.

developmental biology↗

bioRxiv2022

High-resolution Ribosome Profiling Reveals Translational Selectivity for Transcripts in Bovine Preimplantation Embryo Development

High resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome, polysome- and non-polysome-bound RNA profiles of bovine oocytes (GV and MII stage) and early embryos at 2-, 8-cell, morula, and blastocyst stage, and revealed four modes of translational selectivity: i. selective translation of non-abundant mRNAs, ii. active, but modest translation of a selection of highly expressed mRNAs, iii. translationally suppressed abundant to moderately abundant mRNAs, and iv. mRNAs associated specifically with monosomes. A strong translational selection of low abundance mRNAs encoding protein components involved in metabolic pathways and lysosome was found throughout bovine oocyte and preimplantation development. In particular, genes encoding components involved in mitochondrial function were prioritized for translation. Notably, transcripts encoding proteins regulating chromatin modifications selectively translated in oocytes. We found that the translational dynamics largely reflects transcriptional profiles in oocytes and 2-cell embryos, but observed marked shift in translational control in 8-cell embryos associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become better synchronized in morulae and blastocysts. Together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development. Significance StatementTranslational control during preimplantation embryo development is poorly understood, mostly due to the scarcity of samples and the corresponding inability to analyze low quantities of these materials. By developing a low-input method, we have been able to explore the transcriptome, polysome- and non-polysome-bound RNA profiles of bovine oocytes (GV and MII stage) and preimplantation embryos at 2-, 8-cell, morula, and blastocyst stages. We reveal four different modes of translational selectivity, plus novel temporal regulatory mechanisms during early embryo development. The spatiotemporal translation dynamics of bovine oocytes and preimplantation embryos offer an entirely new insight into mammalian embryo development research and new possibilities for improving efficiency of assisted reproduction technologies (ARTs).

developmental biology↗

bioRxiv2021

Tdrd3 regulates the progression of meiosis II through translational control of Emi2 mRNA in mouse oocytes

After completion of meiosis I, the oocyte immediately enters meiosis II and forms a metaphase II (MII) spindle without an interphase, which is fundamental for generating a haploid gamete. Here, we identify tudor domain-containing protein 3 (Tdrd3) as a novel regulator of oocyte meiosis. Although early mitotic inhibitor 2 (Emi2) protein has been shown to ensure the meiosis I to II transition and the subsequent MII spindle formation by inhibiting the anaphase-promoting complex/cyclosome (APC/C), how it accumulates after meiosis I has remained unresolved. We isolated Tdrd3 as a protein directly binding to Emi2 mRNA. In GV-stage mouse oocytes, Emi2 mRNA assembled into RNA granules containing Tdrd3, while cyclin B1 mRNA, which was translated in early meiosis I, formed different granules. Knockdown of Tdrd3 attenuated Emi2 synthesis in meiosis II without affecting cyclin B1 synthesis in meiosis I. Moreover, Tdrd3-deficient oocytes entered interphase and failed to form an MII spindle after completion of meiosis I. Taken together, our results indicate the importance of Tdrd3-mediated translational control of Emi2 mRNA, which promotes Emi2 synthesis in meiosis II, for the progression of meiosis.

developmental biology↗

bioRxiv2020

p38-MAPK mediated rRNA processing and translation regulation enables PrE differentiation during mouse blastocyst maturation.

Backgroundp38-MAPKs are stress-activated kinases necessary for placental development and nutrient and oxygen transfer during murine post-implantation development. In preimplantation development, p38-MAPK activity is required for blastocyst formation. Additionally, we have previously reported its role in regulating specification of inner cell mass (ICM) towards primitive endoderm (PrE), although a comprehensive mechanistic understanding is currently limited. Adopting live embryo imaging, proteomic and transcriptomic approaches, we report experimental data that directly address this deficit. ResultsChemical inhibition of p38-MAPK activity during blastocyst maturation causes impaired blastocyst cavity expansion, most evident between the third and tenth hours post inhibition onset. We identify an overlapping minimal early blastocyst maturation window of p38-MAPKi inhibition (p38-MAPKi) sensitivity, that is sufficient to impair PrE cell fate by the late blastocyst (E4.5) stage. Comparative proteomic analyses reveal substantial downregulation of ribosomal proteins, the mRNA transcripts of which are also significantly upregulated. Ontological analysis of the differentially expressed transcriptome during this developmental period reveals "translation" related gene transcripts as being most significantly, yet transiently, affected by p38-MAPKi. Moreover, combined assays consistently report concomitant reductions in de novo translation that are associated with accumulation of unprocessed rRNA precursors. Using a phosphoproteomic approach, {+/-} p38-MAPKi, we identified Mybpp1a, an rRNA transcription and processing regulator gene, as a potential p38-MAPK effector. We report that siRNA mediated clonal knockdown of Mybpp1a is associated with significantly diminished PrE contribution. Lastly, we show that defective PrE specification caused by p38-MAPKi (but not MEK/ERK signalling inhibition) can be partially rescued by activating the archetypal mTOR mediated translation regulatory pathway. ConclusionsActivated p38-MAPK controls blastocyst maturation in an early and distinctly transient developmental window by regulating gene functionalities related to translation, that creates a permissive environment for appropriate specification of ICM cell fate.

developmental biology↗

bioRxiv2020

The H3.3 chaperone Hira complex orchestrates oocyte developmental competence

Reproductive success relies on a healthy oocyte competent for fertilisation and capable of sustaining early embryo development. By the end of oogenesis, the oocyte is characterised by a transcriptionally silenced state, but the significance of this state and how it is achieved remains poorly understood. Histone H3.3, one of the H3 variants, has unique functions in chromatin structure and gene expression that are cell cycle-independent. We report here a comprehensive characterisation of the roles of the subunits of the Hira complex (i.e. Hira, Cabin1 and Ubn1), which is primarily responsible for H3.3 deposition during mouse oocyte development. Loss-of-function of any component of the Hira complex led to early embryogenesis failure. Transcriptome and nascent RNA analyses revealed that mutant oocytes fail to silence global transcription. Hira complex mutants are unable to establish the H3K4me3 and H3K9me3 repressive marks, resulting in aberrant chromatin accessibility. Among the misregulated genes in mutant oocytes is Zscan4, a 2-cell specific gene that is involved in zygote genome activation. Overexpression of Zscan4 recapitulates the phenotypes of Hira mutants, illustrating that temporal and spatial expression of Zscan4 is fine-tuned at the oocyte-to-embryo transition. Thus, the H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and early embryogenesis by modulating chromatin condensation and transcriptional quiescence.

developmental biology↗

bioRxiv2019

Wwc2 is a novel mitotic/meiotic cell-cycle regulator and cell fate related gene, during preimplantation mouse embryo development and oogenesis

Formation of a mature and hatching mouse blastocyst marks the end of the preimplantation development, whereby regulated cell cleavages culminate in the formation of three distinct lineages. We report dysregulated expression of Wwc2, an ill-characterised paralog of the Hippo-signalling activator Kibra/Wwc1, is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities, typified by imbalanced daughter cell chromatin segregation. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the fertilisation competent stage of meiosis II metaphase arrest, characterised by spindle defects and failed Aurora-A kinase (AURKA) activation. Such cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal dysregulation implicates Wwc2 in maintaining the pluripotent late blastocyst stage epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell-fate.

developmental biology↗

bioRxiv2019

p38-mitogen activated kinases mediate a developmental regulatory response to amino acid depletion and associated oxidative stress in mouse blastocyst embryos

Maternal starvation coincident with preimplantation development has profound consequences for placental-foetal development, with various identified pathologies persisting/manifest in adulthood; the Developmental Origin of Health and Disease (DOHaD) hypothesis/model. Despite evidence describing DOHaD-related incidence, supporting mechanistic and molecular data relating to preimplantation embryos themselves are comparatively meagre. We recently identified the classically recognised stress-related p38-mitogen activated kinases (p38-MAPK) as regulating formation of the extraembryonic primitive endoderm (PrE) lineage within mouse blastocyst inner cell mass (ICM). Thus, we wanted to assay if PrE differentiation is sensitive to amino acid availability, in a manner regulated by p38-MAPK. Although blastocysts appropriately mature, without developmental/morphological or cell fate defects, irrespective of amino acid supplementation status, we found the extent of p38-MAPK inhibition induced phenotypes was more severe in the absence of amino acid supplementation. Specifically, both PrE and epiblast (EPI) ICM progenitor populations remained unspecified and there were fewer cells and smaller blastocyst cavities. Such phenotypes could be ameliorated, to resemble those observed in groups supplemented with amino acids, by addition of the anti-oxidant NAC (N-acetyl-cysteine), although PrE differentiation deficits remained. Therefore, p38-MAPK performs a hitherto unrecognised homeostatic early developmental regulatory role (in addition to direct specification of PrE), by buffering blastocyst cell number and ICM cell lineage specification (relating to EPI) in response to amino acid availability, partly by counteracting induced oxidative stress; with clear implications for the DOHaD model.

developmental biology↗