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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

Schematic diagram and localization of human RPS17.(A) Schematic depiction of RPS17 with a blowout showing the amino acids implicated via NLS mapper software. Eukaryotic linear motif (ELM NLSs) predictions are underlined for the bipartite NLS and shaded for the monopartite NLS. (B) Immunofluorescent staining of Huh7 human liver cells with anti-RPS17 antibody (Top) or with no primary antibody (Bottom) observed with confocal microscopy. Fluorescent intensity in DAPI and Alex594 channels was quantified along a yellow line through one representative cell (Quantification) from both anti-RPS17 antibody or no primary antibody stained cells showing RPS17 signal (red line) with respect to the cytoplasm, nucleus, and nucleolus (blue line). RPS17 localized predominantly to punctate spots within the nucleus as evidenced by the ALEXA594 staining overlaid with DAPI and brightfield images. There was minor nonspecific staining of the anti-mouse Alexa594 antibody within the cytoplasm but none detected in the nucleus suggesting the staining was specific. Small white arrows correspond to the same pair of nucleoli in each panel and were used for the representative quantification. Scale bars on Alexa594 images represent 10 μm.
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pone.0124396.g001: Schematic diagram and localization of human RPS17.(A) Schematic depiction of RPS17 with a blowout showing the amino acids implicated via NLS mapper software. Eukaryotic linear motif (ELM NLSs) predictions are underlined for the bipartite NLS and shaded for the monopartite NLS. (B) Immunofluorescent staining of Huh7 human liver cells with anti-RPS17 antibody (Top) or with no primary antibody (Bottom) observed with confocal microscopy. Fluorescent intensity in DAPI and Alex594 channels was quantified along a yellow line through one representative cell (Quantification) from both anti-RPS17 antibody or no primary antibody stained cells showing RPS17 signal (red line) with respect to the cytoplasm, nucleus, and nucleolus (blue line). RPS17 localized predominantly to punctate spots within the nucleus as evidenced by the ALEXA594 staining overlaid with DAPI and brightfield images. There was minor nonspecific staining of the anti-mouse Alexa594 antibody within the cytoplasm but none detected in the nucleus suggesting the staining was specific. Small white arrows correspond to the same pair of nucleoli in each panel and were used for the representative quantification. Scale bars on Alexa594 images represent 10 μm.

Mentions: Two independent prediction methods were used to analyze the amino acid sequence from the human RPS17 protein. NLS mapper [32] predicted two bipartite NLSs: the first predicted motif occurred between amino acids 3 through 49 (probability score 5.5), and the second predicted motif occurred within amino acids 27 through 49 (probability score 6.1). Eukaryotic linear motif (ELM) analysis [33] also predicted two NLSs within the RPS17. The first ELM-predicted NLS is a bipartite NLS within amino acids 32 through 48 (KRVCEEIAIIPSKKLRN), and the second NLS is predicted to be a monopartite NLS encompassing amino acids 42 to 48 (PSKKLRN) (Fig 1A).


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

Kenney SP, Meng XJ - PLoS ONE (2015)

Schematic diagram and localization of human RPS17.(A) Schematic depiction of RPS17 with a blowout showing the amino acids implicated via NLS mapper software. Eukaryotic linear motif (ELM NLSs) predictions are underlined for the bipartite NLS and shaded for the monopartite NLS. (B) Immunofluorescent staining of Huh7 human liver cells with anti-RPS17 antibody (Top) or with no primary antibody (Bottom) observed with confocal microscopy. Fluorescent intensity in DAPI and Alex594 channels was quantified along a yellow line through one representative cell (Quantification) from both anti-RPS17 antibody or no primary antibody stained cells showing RPS17 signal (red line) with respect to the cytoplasm, nucleus, and nucleolus (blue line). RPS17 localized predominantly to punctate spots within the nucleus as evidenced by the ALEXA594 staining overlaid with DAPI and brightfield images. There was minor nonspecific staining of the anti-mouse Alexa594 antibody within the cytoplasm but none detected in the nucleus suggesting the staining was specific. Small white arrows correspond to the same pair of nucleoli in each panel and were used for the representative quantification. Scale bars on Alexa594 images represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124396.g001: Schematic diagram and localization of human RPS17.(A) Schematic depiction of RPS17 with a blowout showing the amino acids implicated via NLS mapper software. Eukaryotic linear motif (ELM NLSs) predictions are underlined for the bipartite NLS and shaded for the monopartite NLS. (B) Immunofluorescent staining of Huh7 human liver cells with anti-RPS17 antibody (Top) or with no primary antibody (Bottom) observed with confocal microscopy. Fluorescent intensity in DAPI and Alex594 channels was quantified along a yellow line through one representative cell (Quantification) from both anti-RPS17 antibody or no primary antibody stained cells showing RPS17 signal (red line) with respect to the cytoplasm, nucleus, and nucleolus (blue line). RPS17 localized predominantly to punctate spots within the nucleus as evidenced by the ALEXA594 staining overlaid with DAPI and brightfield images. There was minor nonspecific staining of the anti-mouse Alexa594 antibody within the cytoplasm but none detected in the nucleus suggesting the staining was specific. Small white arrows correspond to the same pair of nucleoli in each panel and were used for the representative quantification. Scale bars on Alexa594 images represent 10 μm.
Mentions: Two independent prediction methods were used to analyze the amino acid sequence from the human RPS17 protein. NLS mapper [32] predicted two bipartite NLSs: the first predicted motif occurred between amino acids 3 through 49 (probability score 5.5), and the second predicted motif occurred within amino acids 27 through 49 (probability score 6.1). Eukaryotic linear motif (ELM) analysis [33] also predicted two NLSs within the RPS17. The first ELM-predicted NLS is a bipartite NLS within amino acids 32 through 48 (KRVCEEIAIIPSKKLRN), and the second NLS is predicted to be a monopartite NLS encompassing amino acids 42 to 48 (PSKKLRN) (Fig 1A).

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