Multiple regions of NSR1 are sufficient for accumulation of a fusion protein within the nucleolus.
NSR1, a 67-kD nucleolar protein, was originally identified in our laboratory as a nuclear localization signal binding protein, and has subsequently been found to be involved in ribosome biogenesis. NSR1 has three regions: an acidic/serine-rich NH2 terminus, two RNA recognition motifs, and a glycine/arginine-rich COOH terminus. In this study we show that NSR1 itself has a bipartite nuclear localization sequence. Deletion of either basic amino acid stretch results in the mislocation of NSR1 to the cytoplasm. We further demonstrate that either of two regions, the NH2 terminus or both RNA recognition motifs, are sufficient to localize a bacterial protein, beta-galactosidase, to the nucleolus. Intensive deletion analysis has further defined a specific acidic/serine-rich region within the NH2 terminus as necessary for nucleolar accumulation rather than nucleolar targeting. In addition, deletion of either RNA recognition motif or point mutations in one of the RNP consensus octamers results in the mislocalization of a fusion protein within the nucleus. Although the glycine/arginine-rich region in the COOH terminus is not sufficient to bring beta-galactosidase to the nucleolus, our studies show that this domain is necessary for nucleolar accumulation when an RNP consensus octamer in one of the RNA recognition motifs is mutated. Our findings are consistent with the notion that nucleolar localization is a result of the binding interactions of various domains of NSR1 within the nucleolus rather than the presence of a specific nucleolar targeting signal.
Yan
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Department of Biological Sciences, Columbia University, New York 10027.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002458:Cell Fractionation; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion
D005656:Fungal Proteins; C068036:NSR1 protein, S cerevisiae; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins