Dimerization by introducing deletion or point mutations into full-length hSTAU1 or by expressing exogenous `RBD’5 decreased the potential of hSTAU1 to coimmunoprecipitate with hUPF1 thereby decreasing the efficiency of SMD. Remarkably, inhibiting SMD by disrupting hSTAU1 dimerization promoted keratinocyte-mediated wound-healing, suggesting that dimerization also inhibits the epithelial-to-mesenchymal transition during cancer metastasis.Author Manuscript Author Manuscript Author Manuscript Author Manuscript RESULTSVertebrate STAU includes a conserved motif N-terminal to `RBD’5 Employing yeast two-hybrid analyses, Martel et al.25 demonstrated that full-length hSTAU155 interacts with amino acids 408?96 of a different hSTAU155 molecule. These amino acids consist in the C-terminus of hSTAU155 and contain `RBD’5 (Fig. 1a and Supplementary Fig. 1a), which has only 18 sequence identity towards the prototypical hSTAU1 RBD3 and fails to bind dsRNA15,17. Using ClustalW26, a number of sequence alignments of full-length hSTAU1 with hSTAU2 and STAU orthologs from representatives in the 5 important vertebrate classes revealed a conserved sequence residing N-terminal to `RBD’5 that consists of hSTAU155 amino acids 371?90 (Supplementary Fig.Methyl 5-bromo-3-fluoro-2-methylbenzoate custom synthesis 1a). We get in touch with this motif the Staufen-swapping motif (SSM; Fig. 1a and Supplementary Fig. 1a) for causes explained beneath. In spite of an identifiable `RBD’5, an SSM is absent from, e.g., D. melanogaster or Caenorabditis elegans STAU (Supplementary Fig. 1b). However, STAU in other invertebrates contain each SSM and `RBD’5 regions (Supplementary Fig. 1b). The SSM is proximal towards the TBD, which spans amino acids 282?72 (ref. 15) (Fig. 1a), and it overlaps with amino acids 272?05, at the least part of which recruits hUPF1 during SMD7. Structure of hSTAU1 SSM-`RBD’5 A search with the NCBI Conserved Domain Database27 didn’t recognize hSTAU1 `RBD’5 as an RBD. To know the atomic information of SSM-`RBD’5, we purified hSTAU1 amino acids 367?76 from E. coli (Supplementary Fig. 2a), developed crystals that we verified have been intact employing SDS-polyacrylamide electrophoresis and also silver-staining (Supplementary Fig. 2a), and solved its X-ray crystal structure at 1.7 ?(Table 1). Our structure revealed that `RBD’5 adopts the —- topology of a prototypical RBD and that the SSM types two -helices (hereafter known as SSM 1 and 2) which might be connected by a tight turn (Fig. 1b and Supplementary Fig. 2b). Electron density was clearly interpretable for the SSM and `RBD’5 but not for amino acids 397?02 that constitute the linker (393?06) among SSM and `RBD’5 (Fig.1316852-65-9 manufacturer 1a,b and Supplementary Fig.PMID:27102143 1a). Two conformations had been observed in the Cterminal or `RBD’5 side from the linker, each hinged at L405 so that the position of P404 wasNat Struct Mol Biol. Author manuscript; readily available in PMC 2014 July 14.Gleghorn et al.Pagevariable (Supplementary Fig. 2c). The observed variability raises the possibility that SSM may interact with `RBD’5 as a monomer (cis), dimer (trans), or both inside the crystal structure (Fig. 1b), but we cannot correlate either linker conformation using a monomeric or dimeric state. Each 649 ? interface is developed when the `V’-shape formed by SSM 1 and 2 straddles `RBD’5 1, although the `V’-shape made by `RBD’5 1 and 2 straddles SSM 1 (Fig. 1b ). The intramolecular interactions of an SSM and an `RBD’5 form a core composed of residues with hydrophobic side chains (Fig. 1c). The external solvent boundary of this core is defined by Thr371 on the longer of the two SSM -helic.