Nuclear transport of developmental signaling effectors - PROJECT SUMMARY Heterotaxy, a disorder of left-right development, can be a severe cause of congenital heart disease, which has poor morbidity and mortality even with advancements in surgical and medical management. TGF-β signaling via SMADs is critical to establishing the left-right axis. For SMADs to alter gene transcription and affect embryonic development, they must enter the nucleus. While the mechanism of how SMADs enter the nucleus has been addressed long ago, we feel that a re-examination is necessary. Nuclear transport requires three main elements: 1) Ran dependence, 2) Nuclear Transport Receptor, and 3) NLS (nuclear localization signal) or NES (nuclear export signal) sequences on the cargo. Only recently have we discovered the entire repertoire of NTRs, and in many cases, the tests for requirement and sufficiency for NLS and NES sequences have not been met in SMAD signaling. Part of this logjam is due to the challenges of interrogating the nuclear transport step given the signaling state of the cell, different binding partners, and other functions/interactions with complex signaling networks. We have developed an innovative system using yeast to overcome this barrier. Yeast has a highly conserved nuclear transport system with humans but lacks all of the signaling components of the TGF-β pathway, allowing us to focus on just the nuclear transport step. Exploiting this “minimal system,” we have created an innovative way to test all of the NTRs in vivo using the yeast system and test deletion constructs of the SMADs to localize the NLS/NESs. Finally, our proposal is innovative because of the unique collaboration of developmental biologists, nuclear trafficking experts, and nuclear receptor structural biologists. We propose two Aims: Aim 1 – we will use our yeast system to screen the SMADs for NLS and NES sequences and the critical NTRs that mediate their transport using our innovative rapamycin-inducible NTR depletion strategy and then validate the NTRs for SMAD signaling using F0 CRISPR in Xenopus and test deletion and point mutants for NLS and NES activity using Xenopus functional assays; Aim 2 – we will screen the NTRS for SMAD direct binding and determine the atomic structure of the complex. In this way, each Aim independently screens the SMADs for NLS and NES sequences and relevant NTRs, exploiting the strengths of each system and minimizing the weakness of any one system with redundancy in the others. Finally, with atomic structure, we intend to identify specific interaction sites that dictate NTR binding that could be the target of small molecule disruptors, providing an avenue for therapeutics. Our multidisciplinary collaborative approach should provide synergy within the Aims and create a multi-pronged attack on a fundamental problem in developmental signaling that has important implications for a host of diseases, including cancer.
