Deciphering the overlapping and unique roles of three RNA-binding proteins during oocyte meiosis and early embryogenesis - Oogenesis is a remarkable and precisely regulated process that produces a developmentally competent egg, poised to give rise to an embryo upon fertilization. Central to oogenesis is meiosis, a specialized cell division that halves the chromosome number to generate a haploid egg. During meiosis, transcription is silenced, creating a critical reliance on post-transcriptional control of gene expression to drive cell cycle progression. RNA-binding proteins (RBPs) are important regulators in this context, modulating translation of maternal mRNAs to coordinate the complex events of polar body extrusion, chromosome segregation, and arrest of the egg at meiosis II. Following fertilization, the egg resumes meiosis and completes meiosis II, after which the two pronuclei fuse and undergo nuclear reprogramming essential for early embryonic development. Disruption of translational controls can lead to devastating consequences, including embryo inviability and pregnancy loss. Investigating how RBPs orchestrate translation during meiosis is crucial for understanding factors contributing to early embryo loss. This proposal investigates how the RBPs PATL2, PUM1, and PUM2 regulate translational control to coordinate meiotic and early embryonic cell cycle events. These RBPs have established roles in meiosis, with PATL2 variants linked to oocyte maturation deficiency and early embryonic loss. Using the mouse as a mammalian model, this study leverages cutting-edge technologies to address critical mechanistic questions. The rationale is to define the unique and shared mRNA targets of PATL2, PUM1, and PUM2 and determine how their spatial localization and target specificity shift during the cell cycle and in the absence of other RBPs. Given the conservation of these processes, findings are expected to reveal mechanisms of translational regulation relevant to human oogenesis. This research is driven by two aims: 1) Adapt the HyperTRIBE technique to identify direct mRNA targets of PATL2, PUM1, and PUM2 during oogenesis and early embryogenesis, and assess how loss of one RBP modifies the target prolile of the others; and, 2) Employ spatial transcriptomics to elucidate the spatiotemporal dynamics of translation in individual oocytes and early embryos and assess how these dynamics are disrupted by loss of PATL2, PUM1, or PUM2. By integrating advanced imaging, spatial single-cell RNA sequencing and spatial ribosome profiling with in vitro oocyte maturation and embryo culturing, we will map unique and overlapping targets, localize transcripts in space and time, and reveal changes in target specificity and localization upon RBP depletion. This innovative combination of cutting-edge technologies will enable new insights into translational control essential for producing developmentally competent eggs and embryos. The significance of this work lies in its potential to uncover fundamental principles underlying translational regulation in meiosis, advancing our understanding of infertility and early pregnancy loss and identifying potential therapeutic targets.
