The translocation of DUX4 and DUX4c during myoblast differentiation allows their association with nucleo-cytoplasmic proteins associated with mRNP granules. (2016)

1Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium 2 Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, Columbus, OH, USA 3Laboratory of Proteomic and Microbiology, Research Institute for Biosciences, University of Mons, Mons, Belgium 4Pediatric Department, CHRU Montpellier, Montpellier, France 5I.R.I.B.H.M., Free University of Brussels, Belgium 6Laboratory of Physiology and Experimental Medicine, INSERM U1046, Montpellier, France 7Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH, USA ¶ These authors contributed equally as second authors. & These authors contributed equally as senior authors. Hundreds of double homeobox (DUX) genes map within dispersed 3.3-kb repeated elements and encode DNA-binding proteins. Among these, we identified DUX4 and DUX4c (identical to DUX4 except for the end of the carboxyl terminal domain) which are induced in FSHD muscles. In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4 and DUX4c. Unexpectedly, we identified and validated (by co-IP, GST pull-down and in situ Proximal Ligation Assay) interaction of DUX4 or DUX4c with type III intermediate filament desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomeres at the Z-discs, connect them to sarcolemma proteins, interact with mitochondria, and also contact the nuclear lamina and contribute to positioning of the nuclei. Nuclei and mitochondria positioning are altered in FSHD muscles. Another Z-disc protein, LMCD1, was also validated as a DUX4 partner. We also validated (by co-IP or in situ PLA) several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). The functionality of DUX4-DUX4c interactions with cytoplasmic proteins is underscored by the observation of DUX4/DUX4c nucleo-cytoplasmic translocation upon myoblast fusion and their co-localization with some cytoplasmic DAPI-positive spots. Several validated or identified DUX4/4c partners are also contained in mRNP-granules such as C1QBP (which associates with SRSF9), NONO (which associates with SFPQ), IGF2BP1, IGF2BP3, DHX9 (an ATP-dependent RNA helicase A), ELAVL1, HNRNPU, HNRNPH1, HNRNPQ, PABPC1, PABPC4, PCBP2, nucleolin and several ribosomal proteins (Protein-protein interaction databases at UniProtKB). These complexes contain untranslated mRNAs, travel along microtubules and bring mRNAs to subcellular areas (such as the plasma membrane), where their translation is required at specific times in muscle differentiation. Interestingly, we sometimes observed DUX4 at the cell membrane. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. We observed DUX proteins associated with similar nuclear buds following DUX4 or DUX4c overexpression. In conclusion, in addition to their transcriptional activities, DUX4 and DUX4c overexpression in FSHD muscle cells might disturb cytoskeletal dynamics and mRNA splicing, location and translation by their association with proteins regulating these processes. Further investigations are on-going to confirm such a function that could be common to hundreds of DUX proteins