PROJECT SUMMARY Successful establishment of pregnancy requires the uterus to undergo several well-timed cellular changes to allow the embryo to implant. Thus, even when the blastocyst develops normally, impaired uterine function can lead to implantation failure or early embryo miscarriage. The uterine endometrium prepares for implantation in two steps. First, the endometrial epithelium proliferates, loses polarity, and differentiates, allowing the embryo to attach. Second, the underlying stromal cells proliferate and differentiate into decidual cells, allowing the embryo to implant. These two processes are coordinated by cell-type specific responses to the steroid hormones estrogen and progesterone. However, we lack a complete picture of the downstream responses to these hormones, hampering our ability to develop new strategies to prevent early pregnancy loss. To address this knowledge gap, this proposal focuses on a new area in endometrial physiology, alternative mRNA splicing. Specifically, this proposal will test the central hypothesis that the splicing factor SF3B1 mediates progesterone- driven alternative splicing that is essential for uterine receptivity and decidualization. This idea is founded on the following pieces of preliminary data. First, a high-throughput siRNA screen revealed that SF3B1 was required for human endometrial stromal cell decidualization. Second, knock down of SF3B1 impaired in vitro decidualization more than knock down of eight other splicing factors. Third, treatment with the SF3B1-specific inhibitor Pladienolide B inhibited human endometrial stromal cell decidualization in vitro and murine endometrial decidualization in vivo. Fourth, treatment with Pladienolide B impaired embryo implantation and decidualization in mice. Fifth, SF3B1 protein is elevated in endometrial stromal cells during peri-implantation in mice. Finally, SF3B1 protein but not mRNA in stromal cells was elevated during artificial decidualization in mice, and progesterone stabilized SF3B1 protein but not mRNA in primary human endometrial stromal cells. The work proposed here will build on these strong preliminary data and test the hypothesis by pursuing the following specific aims: (Aim 1) Define the functions of SF3B1 in uterine receptivity and decidualization; (Aim 2) Identify progesterone-induced, SF3B1-dependent alternative splice variants in the endometrium; (Aim 3) Determine the mechanism by which progesterone regulates SF3B1. At the level of basic science, this project will identify the mechanisms that underlie SF3B1-driven mRNA splicing, which is crucial for progesterone- driven endometrial decidualization. Of translational significance, this work will identify novel transcript variants that may contribute to recurrent pregnancy loss. In the long term, such knowledge can be used to develop new strategies to diagnose or prevent early pregnancy loss. Together, this work will help advance Theme 2 of the NICHD 2020 Strategic Plan, which aims to "...