In humans, the phenomenon of cellular reprogramming occurs naturally in a single cell type: the germ cell. At fertilization, the haploid nuclei of a sperm and egg fuse and are reprogrammed by the cytoplasm of the mature oocyte to form a totipotent zygote. Amazingly, this process occurs in the absence of RNA transcription and is inextricably linked to successful spindle assembly and nuclear division during the final stages of meiosis. Thus, post-transcriptional regulation of the transcriptome is paramount to ensure successful spindle assembly prior to completing the oocyte-to-zygote transition. Recent studies in mouse have revealed a critical role at this stage for endogenous-siRNAs (endo-siRNAs) and the RNA binding protein DAZL (which localizes to the spindle poles) in promoting proper spindle assembly. However, it remains unclear how they cooperate to directly regulate this process. Preliminary data suggest that DAZL and endo-siRNAs both regulate transcripts related to the RanGTP pathway, a critical player in the formation of the mitotic spindle. Therefore, I hypothesize that DAZL and endo-siRNAs work together to spatially regulate translation of target mRNAs that are critical for meiotic spindle organization and cell cycle progression. To address this hypothesis, genetic fusion constructs with various deletions of protein domains within DAZL combined with live oocyte imaging during maturation will be used to determine the mechanism and importance of DAZL localization to the meiotic spindle. In addition, mRNA targets of DAZL will be immunoprecipitated and studied to determine the downstream effects of DAZL's role on formation of the meiotic spindle, particularly with regards to the levels of RanGTP. Finally, the connection between DAZL and endo-siRNAs will be explored through their connection to the RanGTP pathway. Various luciferase constructs, FRET probes, knockdown and overexpression assays, as well as small RNA-deficient (Dicer mutant) oocytes will be used to determine whether endo-siRNAs directly regulate Rangap1, and thus RanGTP levels, to facilitate normal spindle assembly. The experiments detailed in this proposal will elucidate how an RNA binding protein, DAZL, and endo-siRNAs work together to coordinately regulate spindle assembly and, thus, cell fate. This work will also provide novel insights for better understanding other pluripotent stem cell populations, including the induced reprogramming of somatic cells to pluripotent cells.
The regulation of germ cell cycle and reprogramming at fertilization, particularly in the oocyte, is crucial to reproductive health. Disruptions of germ cll development lead to sterility, the formation of germ cell tumors, and aberrations during early embryonic development. This work will uncover novel mechanisms that regulate organization and function of the final cell division in oocytes before reprogramming to an embryo after fertilization.
