title: Identification of novel non-canonical RNA-binding sites in Gemin5 involved in internal initiation of translation.
abstract: Ribonucleic acid (RNA)-binding proteins are key players of gene expression control. We have shown that Gemin5 interacts with internal ribosome entry site (IRES) elements and modulates initiation of translation. However, little is known about the RNA-binding sites of this protein. Here we show that the C-terminal region of Gemin5 bears two non-canonical bipartite RNA-binding sites, encompassing amino acids 1297-1412 (RBS1) and 1383-1508 (RBS2). While RBS1 exhibits greater affinity for RNA than RBS2, it does not affect IRES-dependent translation in G5-depleted cells. In solution, the RBS1 three-dimensional structure behaves as an ensemble of flexible conformations rather than having a defined tertiary structure. However, expression of the polypeptide G51383-1508, bearing the low RNA-binding affinity RBS2, repressed IRES-dependent translation. A comparison of the RNA-binding capacity and translation control properties of constructs expressed in mammalian cells to that of the Gemin5 proteolysis products observed in infected cells reveals that non-repressive products accumulated during infection while the repressor polypeptide is not stable. Taken together, our results define the low affinity RNA-binding site as the minimal element of the protein being able to repress internal initiation of translation.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 1, 28049-Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Fernandez-Chamorro
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 1, 28049-Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Piñeiro
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028-Barcelona, Spain.
authors.forename: James M B
authors.initials: JM
authors.lastname: Gordon
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 1, 28049-Madrid, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Ramajo
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 1, 28049-Madrid, Spain.
authors.forename: Rosario
authors.initials: R
authors.lastname: Francisco-Velilla
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028-Barcelona, Spain Catalan Institution for Research and Advanced Studies (ICREA), Passeig Lluis Companys 23, 08010-Barcelona, Spain.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Macias
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 1, 28049-Madrid, Spain emartinez@cbm.csic.es.
authors.forename: Encarnación
authors.initials: E
authors.lastname: Martinez-Salas
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D055532:SMN Complex Proteins
chemical_list: C449808:GEMIN5 protein, human; D012333:RNA, Messenger; D012367:RNA, Viral; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins