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Identification and fine mapping of nuclear and nucleolar localization signals within the human ribosomal protein S17.

Kenney SP, Meng XJ - PLoS ONE (2015)

Bottom Line: Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70.Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17.The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University (Virginia Tech), Blacksburg, Virginia, United States of America.

ABSTRACT
Human ribosomal protein S17 (RPS17) is mutated in Diamond-Blackfan Anemia (DBA), a bone marrow disorder that fails to produce sufficient red blood cells leading to anemia. Recently, an RPS17 protein sequence was also found to be naturally inserted in the genome of hepatitis E virus (HEV) from patients chronically-infected by HEV. The role of RPS17 in HEV replication and pathogenesis remains unknown due to the lack of knowledge about how RPS17 functions at a molecular level. Understanding the biological function of RPS17 is critical for elucidating its role in virus infection and DBA disease processes. In this study we probed the subcellular distribution of normal and mutant RPS17 proteins in a human liver cell line (Huh7). RPS17 was primarily detected within the nucleus, and more specifically within the nucleoli. Using a transient expression system in which RPS17 or truncations were expressed as fusions with enhanced yellow fluorescent protein (eYFP), we were able to identify and map, for the first time, two separate nuclear localization signals (NLSs), one to the first 13 amino acids of the amino-terminus of RPS17 and the other within amino acids 30-60. Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70. Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17. The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes.

No MeSH data available.


Related in: MedlinePlus

Trafficking of triple eYFP RPS17 protein truncations.(A) Schematic depicting RPS17 protein and tested truncations along with a summary of their cellular localization (Right). Nuc: nuclear; Cyt: cytoplasmic. NLS mapper predictions are represented by lines above the full-length protein, ELM NLS predictions are indicated by the shading within the full-length RPS17 representation. (B) Confocal microscopy images of cells expressing the indicated amino acids fused to triple eYFP listed in white. Amino acids 51–135, 12–40, 39–51 were not capable of actively trafficking triple eYFP to the nucleus whereas 1–13, 27–66, 43–61, and 32–50 were capable of trafficking triple eYFP into the nucleus. Scale bars on merged images represent 10 μm.
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pone.0124396.g003: Trafficking of triple eYFP RPS17 protein truncations.(A) Schematic depicting RPS17 protein and tested truncations along with a summary of their cellular localization (Right). Nuc: nuclear; Cyt: cytoplasmic. NLS mapper predictions are represented by lines above the full-length protein, ELM NLS predictions are indicated by the shading within the full-length RPS17 representation. (B) Confocal microscopy images of cells expressing the indicated amino acids fused to triple eYFP listed in white. Amino acids 51–135, 12–40, 39–51 were not capable of actively trafficking triple eYFP to the nucleus whereas 1–13, 27–66, 43–61, and 32–50 were capable of trafficking triple eYFP into the nucleus. Scale bars on merged images represent 10 μm.

Mentions: To define the specific amino acid sequences required for nuclear import, varying lengths of RPS17 were cloned in frame with the triple eYFP protein allowing us to observe subcellular localization (Fig 3A). RPS17 amino acids 51 through 135 attached to triple eYFP remained cytoplasmic (Fig 3B panel c), suggesting that no nuclear trafficking motifs were present within the C-terminal 84 amino acids of RPS17. Amino acids 1–38 and 27–66 each resulted in nuclear localization of the triple eYFP protein, suggesting the existence of either an NLS within the overlapping amino acids 27–38 or multiple nuclear localization signals within the first 38 amino acids of RPS17 and within amino acids 27–66 (Fig 3B panels a and e respectively).


Identification and fine mapping of nuclear and nucleolar localization signals within the human ribosomal protein S17.

Kenney SP, Meng XJ - PLoS ONE (2015)

Trafficking of triple eYFP RPS17 protein truncations.(A) Schematic depicting RPS17 protein and tested truncations along with a summary of their cellular localization (Right). Nuc: nuclear; Cyt: cytoplasmic. NLS mapper predictions are represented by lines above the full-length protein, ELM NLS predictions are indicated by the shading within the full-length RPS17 representation. (B) Confocal microscopy images of cells expressing the indicated amino acids fused to triple eYFP listed in white. Amino acids 51–135, 12–40, 39–51 were not capable of actively trafficking triple eYFP to the nucleus whereas 1–13, 27–66, 43–61, and 32–50 were capable of trafficking triple eYFP into the nucleus. Scale bars on merged images represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390217&req=5

pone.0124396.g003: Trafficking of triple eYFP RPS17 protein truncations.(A) Schematic depicting RPS17 protein and tested truncations along with a summary of their cellular localization (Right). Nuc: nuclear; Cyt: cytoplasmic. NLS mapper predictions are represented by lines above the full-length protein, ELM NLS predictions are indicated by the shading within the full-length RPS17 representation. (B) Confocal microscopy images of cells expressing the indicated amino acids fused to triple eYFP listed in white. Amino acids 51–135, 12–40, 39–51 were not capable of actively trafficking triple eYFP to the nucleus whereas 1–13, 27–66, 43–61, and 32–50 were capable of trafficking triple eYFP into the nucleus. Scale bars on merged images represent 10 μm.
Mentions: To define the specific amino acid sequences required for nuclear import, varying lengths of RPS17 were cloned in frame with the triple eYFP protein allowing us to observe subcellular localization (Fig 3A). RPS17 amino acids 51 through 135 attached to triple eYFP remained cytoplasmic (Fig 3B panel c), suggesting that no nuclear trafficking motifs were present within the C-terminal 84 amino acids of RPS17. Amino acids 1–38 and 27–66 each resulted in nuclear localization of the triple eYFP protein, suggesting the existence of either an NLS within the overlapping amino acids 27–38 or multiple nuclear localization signals within the first 38 amino acids of RPS17 and within amino acids 27–66 (Fig 3B panels a and e respectively).

Bottom Line: Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70.Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17.The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University (Virginia Tech), Blacksburg, Virginia, United States of America.

ABSTRACT
Human ribosomal protein S17 (RPS17) is mutated in Diamond-Blackfan Anemia (DBA), a bone marrow disorder that fails to produce sufficient red blood cells leading to anemia. Recently, an RPS17 protein sequence was also found to be naturally inserted in the genome of hepatitis E virus (HEV) from patients chronically-infected by HEV. The role of RPS17 in HEV replication and pathogenesis remains unknown due to the lack of knowledge about how RPS17 functions at a molecular level. Understanding the biological function of RPS17 is critical for elucidating its role in virus infection and DBA disease processes. In this study we probed the subcellular distribution of normal and mutant RPS17 proteins in a human liver cell line (Huh7). RPS17 was primarily detected within the nucleus, and more specifically within the nucleoli. Using a transient expression system in which RPS17 or truncations were expressed as fusions with enhanced yellow fluorescent protein (eYFP), we were able to identify and map, for the first time, two separate nuclear localization signals (NLSs), one to the first 13 amino acids of the amino-terminus of RPS17 and the other within amino acids 30-60. Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70. Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17. The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes.

No MeSH data available.


Related in: MedlinePlus