Deciphering molecular functions of the “multitalented” scaffold protein USH1G/SANS in primary cilia and the nucleus

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SANS (scaffold protein containing ankyrin repeats and SAM domain) is encoded by USH1G gene and serves as a central scaffold molecule of the protein interactome related to the human Usher syndrome (USH), the most common form of heritetery combined deaf-blindness. Although defects in USH1G/SANS predominately lead to dysfunctions in the eye and the inner ear in patients, it is almost ubiquitously expressed. Previous studies have demonstrated that the scaffold protein participates in diverse cell modules and processes, such as the assembly of mechanosensitive signaling complexes, primary ciliogenesis, intracellular transport, endocytosis, and pre-mRNA splicing. Here, we will focus on molecular interactions and the dynamics of processes related to SANS in primary cilia and the nucleus. In our project, we aim to unravel the molecular function of USH1G/SANS in primary cilia and the nucleus by integrative approaches, combining different methods from biochemistry, molecular cell biology and biophysics, as well as molecular modeling. We will decipher the interactions with ligand proteins and their binding kinetics and properties to SANS, monitor the molecular dynamics of MLOs and between them in the living cell, and the LLPS properties in the MLOs of both cell organelles, namely the primary ciliary base and the nuclear speckles or Cajal bodies in the nucleus. We have the following specific objectives: (1) Unraveling the physico-chemical basis for multivalent protein binding to SANS. (2) Elucidation the function of SANS for the molecular dynamics in the nucleus and primary cilia. (3) Evaluation of SANS-dependent molecular interconnections between nucleus and primary cilia. (4) Deciphering the role of SANS in the formation of polymer condensates by LLPS. With our experimental approaches and the in-silico modeling we aim to deliver a comprehensive view of the molecular mechanisms underlying the role of SANS in protein-protein interaction and LLPS. We expect not only novel insights into common mechanisms and the dynamics and interplay of LLPS, but also further understanding of the regulation of the spliceosome and ciliogenesis and /or deciliation. In addition, prospective results will elucidate the link between nuclear and ciliary MLOs. Moreover, we expect to pinpoint to what extent alterations in the formation of LLPS and in the “interMLOar” exchange are disease-relevant, underlying the pathophysiology of human Usher syndrome.