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

Mammalian expression cytomegalovirus promoter-based vector constructs encoding N-terminal tagged Flag-NOA36 and truncated Flag-N34-NOA36 (Invitrogen), C-terminal tagged NOA36-eGFP (Clontech) were generated by PCR amplification from the human full-length NOA36 cDNA. PCR products incorporated EcoRI and the BamHI sites on the products ends. A truncated fusion protein containing amino acids 1-33 of NOA36 was also expressed as eGFP fusion protein. Site-directed mutagenesis was carried out on this p1-33-NOA-eGFP truncated protein to generate deletion of several amino acids, or alternatively, to convert a basic amino acid to a neutral amino acid. The sequence data for the mutagenic oligonucleotides are shown in Table S1 . The entire coding region of the yeast exopolyphosphatase, scPPX1, was obtained from the plasmid pTrcPPX1 [41] , which was kindly provided by the late Prof. Arthur Kornberg, Stanford University School of Medicine (Stanford, CA). Coding sequences for scPPX1 and for the residues 1-33 of NOA36 were ligated to a vector pcDNA3.1 (Invitrogen) that had previously been modified to contain a Flag epitope. The sequence for the resultant recombinant protein, Flag-1-33-NOA36-scPPX1, was further subcloned into the eukaryotic expression vector pIRES2-EGFP Table 1 . Sequences of several nucleolar localization signals in which one or more clusters of basic amino acids are present.