Molecular mechanism of Vasa function in localized mRNA translation on the spindle - PROJECT SUMMARY Localization of mRNA and its on-site translation (localized translation) enables spatiotemporal control of protein synthesis in the cell. The mitotic spindle is a sub-cellular region where localized translation has been reported to occur, yet its mechanism of regulation is largely unknown in any model system. We previously reported a conserved DEAD-box RNA helicase, Vasa, as a promising factor that regulates localized translation on the spindle in the sea urchin embryo. At asymmetric cell division, Vasa asymmetrically localizes on one side of the spindle, which is critical for asymmetric translation and the formation of micromeres, a major signaling center in the embryo. The preliminary results suggest that the predicted Vasa's target mRNAs are maternally loaded. Further, transcription is scarce during early embryogenesis. Therefore, we hypothesize that Vasa recruits and unlocks the maternal mRNAs for translation on the spindle, providing spatiotemporal control in protein synthesis during embryogenesis. To test this hypothesis, Question 1 will identify Vasa's target mRNAs and their functional contributions to mitosis. As a preliminary study, we computationally predicted several of Vasa's target mRNAs, which all appear to be involved in mitosis. We will identify how Vasa is critical for these targets' localized translation on the spindle and how they contribute to mitosis by real-time imaging of translation in developing embryos. Further, we will perform APEX-seq to comprehensively identify Vasa's target mRNAs on the spindle, followed by experimental validations of the obtained data. Question 2 will reveal the significance of Vasa-mediated asymmetric translation to embryonic development. Using optogenetics, we will deplete Vasa specifically at the 16-cell stage when micromeres are formed. We will process the resultant embryos at the 16-cell, morula, and blastula stages for single- cell (sc)RNA-seq, followed by experimental validations of the obtained data. The resultant data will identify what mRNAs Vasa asymmetrically recruits and translates in micromeres at the 16-cell stage and how its depletion blocks micromeres' signaling function, casing developmental defects at later stages. Question 3 will identify how Vasa selects its target mRNAs for localized translation. Preliminary results suggest that Vasa may recruit mRNAs with Guanine quadruplex (G4) secondary structure. Using both endogenous and synthetic G4-mRNA constructs, we will determine the essentiality of the G4 motif for Vasa's recruitment by real-time imaging of the mRNA. We will also reveal how the G4 motif impacts the timing and location of Vasa's target mRNA translation by real-time imaging of translation in embryos. Future steps include identifying Vasa's partners responsible for its granule assembly and how that controls its target mRNA's localized translation. We will also integrate the obtained omics data into our current prediction to identify Vasa's conserved targeting mechanism and function across organisms.
