Journal
NUCLEUS
Volume 6, Issue 4, Pages 314-325Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/19491034.2015.1079680
Keywords
fluorescence microscopy; GFP; nucleolus; nucleolar localization sequence; protein targeting
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Funding
- Fundacao para a Ciencia e Tecnologia, Portugal [SFRH-BPD-66611-2009]
- German Research Council [DFG CA198/3]
- Fundação para a Ciência e a Tecnologia [SFRH/BPD/66611/2009] Funding Source: FCT
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The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.
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