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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

Localization of the full-length RPS17 containing point mutations fused N-terminally to single yellow fluorescent protein.(A) A schematic showing the amino acids found to be important for nuclear import in the context of the truncation mutants. Wild-type amino acids are shown above with amino acid number and residues altered to alanine are depicted below. (B) Confocal microscopy images of full-length RPS17 containing the denoted mutations fused with triple eYFP. Scale bars on merged images represent 10 μm.
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pone.0124396.g005: Localization of the full-length RPS17 containing point mutations fused N-terminally to single yellow fluorescent protein.(A) A schematic showing the amino acids found to be important for nuclear import in the context of the truncation mutants. Wild-type amino acids are shown above with amino acid number and residues altered to alanine are depicted below. (B) Confocal microscopy images of full-length RPS17 containing the denoted mutations fused with triple eYFP. Scale bars on merged images represent 10 μm.

Mentions: After identifying the amino acid residues required for nuclear import within the context of RPS17 truncations, we next wanted to determine if these residues were also critical for nuclear import or if other residues played a role in the context of the full-length RPS17 protein. We first mutated lysines 10, 11, 32, 33, 44, and 45 to alanine (Fig 5A) and we could still detect eYFP fluorescence within the nucleus (Fig 5B top). We next mutated lysine 49 to alanine in addition to amino acids 10, 11, 32, 33, 44, and 45 to alanine. This RPS17 mutant was no longer capable of transiting into the nucleus (Fig 5B bottom).


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

Kenney SP, Meng XJ - PLoS ONE (2015)

Localization of the full-length RPS17 containing point mutations fused N-terminally to single yellow fluorescent protein.(A) A schematic showing the amino acids found to be important for nuclear import in the context of the truncation mutants. Wild-type amino acids are shown above with amino acid number and residues altered to alanine are depicted below. (B) Confocal microscopy images of full-length RPS17 containing the denoted mutations fused with triple eYFP. 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.g005: Localization of the full-length RPS17 containing point mutations fused N-terminally to single yellow fluorescent protein.(A) A schematic showing the amino acids found to be important for nuclear import in the context of the truncation mutants. Wild-type amino acids are shown above with amino acid number and residues altered to alanine are depicted below. (B) Confocal microscopy images of full-length RPS17 containing the denoted mutations fused with triple eYFP. Scale bars on merged images represent 10 μm.
Mentions: After identifying the amino acid residues required for nuclear import within the context of RPS17 truncations, we next wanted to determine if these residues were also critical for nuclear import or if other residues played a role in the context of the full-length RPS17 protein. We first mutated lysines 10, 11, 32, 33, 44, and 45 to alanine (Fig 5A) and we could still detect eYFP fluorescence within the nucleus (Fig 5B top). We next mutated lysine 49 to alanine in addition to amino acids 10, 11, 32, 33, 44, and 45 to alanine. This RPS17 mutant was no longer capable of transiting into the nucleus (Fig 5B bottom).

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