Ciliopathies, Usher syndrome, and splicing

1 PhD project offered in the IPP summer call 2020

Scientific Background

The machineries of intrinsic functions of the nucleus are organized in membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LPPS). Pre-mRNA splicing is catalysed by the spliceosome, a highly dynamic macromolecular complex closely associated with nuclear speckles and Cajal bodies, prominent MLOs in the nucleoplasma. There is also growing evidence that membrane-less polymer condensates are the basis for the organization of functional modules in primary cilia, the sensory antenna of the cell. Recent studies demonstrated that, unexpectedly, nuclear proteins are also present in primary cilia and vice versa, ciliary molecules localize to nuclear foci (Johnson & Malicki 2019). As expected, pre-mRNA processing factors are found in the splicing compartments of the nucleus, but we additionally found them at the ciliary base where they promote ciliogenesis (Wheway et al. 2015). More recently we demonstrated that the Usher syndrome USH1G/SANS protein is not only part of ciliary modules, but is also found in the nucleoplasm. In the nucleus, SANS regulates splicing by facilitating the intranuclear transfer of spliseosomal tri-snRNP complexes from Cajal bodies to the nuclear speckles to facilitate the formation of active spliceosome (Yildirim et al. submitted). In our study, we also showed that the absence of SANS leads to significant changes in both splicing related MLOs, an increase in the number and the condensation of polymers in Cajal bodies and spreading of nuclear speckles indicating the loss of LLPS (Yildirim et al. sub.). Defects in SANS and its depletion leads to deffective consitiutive and alternative splicing of specific target genes related to cell proliferation, ciliation and USH.

PhD project

We aim to decipher the roles of SANS in a. molecular mechanisms underlying the release of snRNPs from Cajal bodies and the recruitment to the nuclear speckles and for b. the recruitment and release of molecules to/from the ciliary base. For this we will team up with life and polymer scientists to elucidate the interconnection and dynamics of these modules by an integrative approach combining various interdisciplinary methods, using polymer concepts for the description of the dynamics in the living system. We aim to task the following points: 1. Do the multivalent interactions of SANS drive formation of polymer condensates via LLPS in the nucleus and at the base of primary cilia? For this we will quantitatively study the formation and condensation of SANS-related protein complexes both in vitro and in living cells. 2. How does the intrinsically disordered regions of SANS_CENTn regulate the interaction of SANS with its numerous interaction partners of SANS? Besides NMR (in collaboration), fluorescence resonance energy transfer (FRET) assays, in situ proximity ligation assays (PLAs) and surface plasmon resonance (SPR) will be used as complementary methods. 3. How dynamic are the MLOs under physiological conditions? 4. Are MLOs in the nucleus and MLOs at the ciliary base molecularly and/or functionally linked by the dynamic exchange of components? For 3. and 4., we will apply quantitative live cell imaging applying photoactivatable fluorescent proteins in spinning disk microscopy and 3D stimulated emission depletion (STED) microscopy.
We expect a compressive view of the molecular mechanisms underlying the formation and regulation of MLOs in the nucleus and in primary cilia. Our data will provide not only novel insights into common mechanisms related to the dynamics and interplay of LLPS, but also further understanding of the regulation of the spliceosome and ciliogenesis. From a biomedical point of view, we also anticipate to elucidate the pathophysiology leading to the senso-neuronal degenerations associated with the human Usher syndrome.

Publications relevant to the project

Yildirim A, Mozaffari-Jovin S, Wallisch, A-K, Ries J, Urlaub H, Lührmann R and Wolfrum U (submitted) The ciliopathy protein SANS (USH1G) regulates pre-mRNA splicing by mediating the intranuclear transfer of tri-snRNPs from Cajal bodies to the pre-spliceosome in splicing speckles.

Sorusch N, Yildirim, A, Knapp B, Janson J, Fleck W, Scharf C, and Wolfrum U (2019) SANS (USH1G) molecularly links the human Usher syndrome protein network to the intraflagellar transport module by direct binding to IFT-B proteins.Front Cell Dev Biol. 7:216. eCollection 2019.

Sorusch N, Bauß K, Plutniok J, Samanta A, Knapp B, Nagel-Wolfrum K and Wolfrum U (2017) Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex.Hum Mol Genet. 26(6):1157-1172.

Wheway G, et al. Wolfrum U, Beales PL, Gibson T, Doherty D, Mitchison HM, Roepman R, Johnson CA (2015) An siRNA-based functional genomics screen for the identification of novel regulators of ciliogenesis and ciliopathy genes. Nat Cell Biol 17:1074-87

Bauss K, Knapp B, Jores P, Roepman R, Kremer H, v. Wijk E, Maerker T, Wolfrum U (2014) Phosphorylation of the Usher syndrome protein SANS controls Magi2-mediated endocytosis Hum Mol Genet 23:3923-42.

Contact Details

Uwe Wolfrum