Impact of Gemin5 in protein synthesis: phosphoresidues of the dimerization domain regulate ribosome binding

RNA-binding proteins are involved in all steps of gene expression. Their malfunction has important consequences for cell growth through dysregulation of protein synthesis events leading to cancer. Gemin5 is a predominantly cytoplasmic protein involved in spliceosome assembly and gene expression reprogramming. The protein is phosphorylated at multiple sites, although the role of the individual phosphorylated residues remains poorly understood. With the aim to understand the impact of Gemin5 post-translation modifications for RNA-binding, protein synthesis, and therefore cell growth, we have analysed the role of conserved P-residues located in the dimerization domain of the protein in subcellular localization, protein stability, interactome, ribosome binding and translation regulation. We show that the activation of signalling pathways in response to a dsRNA mimic, which leads to phosphorylation of eIF2α, enhanced the intensity of Gemin5 binding to a cognate RNA ligand. In addition, ribosome binding decreased when Ser/Thr 847 and 852–854 are substituted by a non-phosphorylatable residue, consistent with decreased protein stability, and reduced number of associated factors. Similar analyses of phosphomimetic mutants (S847D and STS852-854DDD) suggested conformational changes of the protein structure as the responsible factor for the defective proteins. Moreover, cap-dependent protein synthesis was significantly altered by the triple substitution STS/DDD, pointing towards a role of these residues in protein synthesis regulation.