NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

To investigate the localization of the different regions of the NOA36 protein, we fused eGFP to three truncated peptides corresponding to the different putative domains deduced from the amino acid sequence: the amino terminal domain which extends from the first methionine to the last conserved basic amino acid before the cysteine-rich domain (amino acids 1 to 33); the central cysteine-rich region (amino acids 34 to 239); and the polyacidic carboxy terminal (amino acids 240 to 320). These constructs were analyzed in transient transfection assays in HeLa cells to determine the subcellular localization of the fusion proteins. Out of the three constructs tested, only the basic rich amino terminal peptide was capable of transporting eGFP to the nucleolus (Fig. 2) . The cysteine-rich domain was distributed all over the cell, and the polyacidic carboxy terminal showed only a cytoplasmic localization. This cytoplasmic localization was similar to that found in the assays with the full-length protein fused to eGFP ( Fig. 2A) . To confirm that it is the amino terminal that is responsible for NOA36 nucleolar localization, we fused the Flag epitope to the amino terminus of a NOA36 truncated protein in which the amino acids 2 to 33 had been deleted. HeLa cells transiently transfected with this construct showed nuclear but not nucleolar staining of the Flag epitope, which was verified by the absence of colocalization with the nucleolar marker UBF in all the interphase transfected cells (Fig. 2C) . Besides the nuclear staining, the 13.6% of the transfectants, also showed a diffuse or punctuated cytoplasmic pattern similar to that observed in the Flag-NOA36 fusion protein.