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 human NOA36 amino terminal sequence contains a high proportion of lysine and arginine residues, with basic amino acids accounting for 40% in the sequence 1 to 33. The analysis of this sequence with Prosite software predicted the existence of a putative bipartite nuclear localization signal (NLS) between amino acids 3-20 [35] . The alignment of the NLS of human NOA36 with other NOA36 sequences from phyla as distant from each other as cnidarians and mammals reveals a remarkable degree of conservation, showing an identity of 66.6% (Fig. 3A) . This peptide contains two clusters of three basic amino acids as well as other residues conserved in all the analyzed NOA36 protein sequences. The consensus sequence in the 20 first amino acids is M-P-K-K-K-S/T-G-A/Q-R-K-K-A-E-N/S/K-R/Q-R/K/N-E/V-R/I-X-K. However, other basic amino acids are present in the sequence 21 to 33 that could be significant for the nucleolar localization. In order to characterize the ability of the NOA36 amino terminal sequence to direct eGFP to the nucleolus in different species, we studied the localization of the recombinant protein 1-33NOA-eGFP by fluorescence microscopy in transient transfected cell cultures from different cell types and species. Our results showed that this peptide is sufficient to direct eGFP to the nucleolus in all the interphase cells in two different human cell lines, HeLa (fig. 2C) and PC3 (Fig. 3B) , and in hamster CHO cells (data not shown). We found that this NoLS has been highly conserved during evolution, since fish and insect cells transfected with this construct also show nucleolar staining (Fig. 3B) . These results are consistent with the high degree of conservation presented by this peptide over the course of evolution.