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

The three clusters of basic amino acids of this NoLS are highly conserved during evolution, since the first two clusters are present in all the available orthologous sequences of NOA36, from cnidarian to mammalian species, while the third cluster is present in all the vertebrate sequences analyzed (Fig. 3A) . We therefore decided to study the effect of deleting each of these clusters in turn (constructs (D3-5) and (m9D10-13)). The analysis of the quantification of these constructs revealed that only the cluster of three lysine residues in positions 3-5 was essential for maintaining the NoLS, since the deletion of these three lysine residues provoked a nuclear distribution of eGFP very similar to that of the empty vector (nucleolus/nucleus ratio = 1.5). This effect was confirmed by a mutant in which these three basic amino acids were changed to neutral amino acids (nucleolus/nucleus ratio = 2.2). The elimination of the second cluster (amino acids 9-11), however, did not show statistically significant differences in fluorescence intensity with respect to the wild type peptide, although it did show a lower nucleolus/nucleus ratio (8.7). Similar results were obtained when an Arg residue was mutated to Ser (m15) in the third cluster (nucleolus/nucleus ratio = 8.1). These data suggest that the main role in the nucleolar localization is played by the first cluster of lysine residues in position 3-5, with the other two clusters of basic amino acids playing a minor role. These data also suggest that the first cluster alone might be sufficient to promote nucleolar transport of the eGFP. In order to answer this question, that is, whether this first cluster is in fact the NoLS of NOA36, we fused amino acids 1-5 to eGFP (construct (D6-33) ). However, the quantification of the relative fluorescence intensity of this construct revealed that, although the nucleolar localization was slightly higher than in the empty vector (nucleolus/nucleus ratio = 2.2), this short peptide did not retain the capability of transporting the eGFP to the nucleus (nucleus/cytoplasm ratio = 3) (Fig. 4C and D) . We also studied the significance of the amino acids not included in the basic amino acids region (amino acids 21 to 33). With this aim, we eliminated this sequence from the reference peptide (construct (D21-33) ). The quantification of fluorescence intensity revealed that there were no statistically significant differences when it was compared with the pN33-eGFP reference construct (nucleolus/ nucleus ratio = 9) (Fig. 4C and 4D) .