Subcellular transport, regulation, and function of RNAs during early development - PROJECT SUMMARY During early development, all animal embryos undergo several rounds of rapid early cleavage divisions under time constraint and exquisite regulation. It is still unclear how such exquisite regulation of mitosis is achieved. We have recently discovered that miRNA-31-mediated local translation at the mitotic spindle is important for early development and highlight the importance of post-transcriptional regulation in ensuring proper mitosis. Localization of miR-31 to the mitotic spindle is evolutionarily conserved, as we observe this localization in both sea urchin embryos and mammalian cells. This discovery has opened up key questions. Namely, how specific RNAs are transported to the mitotic spindle and what are the specific functions of proteins encoded by these localized transcripts. We will use the sea urchin embryo to address key questions in RNA regulation, and mammalian cells to test for evolutionary conservation. The relatively fast early development and experimental manipulability of the sea urchin embryo, along with its well-described gene regulatory networks and some miRNA characterizations, and conserved developmental processes are key features that make it an excellent model. Importantly, the sea urchin embryo provides an organismal-level understanding of conserved mechanism of RNA localization and post-transcriptional control. Our future work will focus on two research themes: (1) We will investigate the molecular mechanism of subcellular transport of RNAs (miRNAs and their targets) to the mitotic spindle in sea urchin embryos and mammalian cells. How RNAs are transported to the subcellular region of mitotic spindle remains unclear. We hypothesize that miRNAs and their target RNAs are co- transported by RNA Binding Proteins as a ribonucleoprotein complex to the mitotic spindle, in part by hitchhiking on organelles. The significance of this study is that by identifying evolutionarily conserved, fundamental mechanisms of subcellular transport of miRNAs and their target RNAs, we will define how spatiotemporal control is achieved. (2) We will identify the regulation and function of these RNAs during the fast dividing phase of the cleavage stage sea urchin embryos and in mammalian cells. We will test the hypothesis that the level of translated miRNA targets needs to be carefully modulated at the mitotic spindle to mediate chromosomal segregation. We will further examine how miR-31 regulates its targets en route to the mitotic spindle, as well as reveal the functions of miR-31 targets that are critical for chromosomal segregation. Our long-term goal is to obtain a comprehensive mechanistic understanding of the subcellular transport of RNA, RNA post-transcriptional regulation mediated by miRNAs, and the functions of proteins they encode during mitosis. Understanding the mechanistic basis of how RNAs are transported to specific subcellular locations and their functional significance in the spatiotemporal context will represent a transformative advance in our knowledge of how an embryo regulates its precise timing of mitosis, the myriad roles RNAs play in early development and disease, in turn impacting the development of RNA-based and RNA-targeted therapies.
