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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

Mapping the determinants of nucleolar localization within RPS17.(A) Schematic depicting amino acids from nuclear localization sequence (NLS) 1 (amino acids 1–13) and NLS 2 (amino acids 32–50) in concert with each other separated via a glycine serine flexible linker or individually in the presence of the di-RG motif (amino acids 50–70). (B) Confocal microscopy images depicting localization of specified RPS17 amino acids attached to triple eYFP protein. Arrows denote the same single nucleoli in each set of panels. Scale bars on merged images represent 10 μm.
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pone.0124396.g006: Mapping the determinants of nucleolar localization within RPS17.(A) Schematic depicting amino acids from nuclear localization sequence (NLS) 1 (amino acids 1–13) and NLS 2 (amino acids 32–50) in concert with each other separated via a glycine serine flexible linker or individually in the presence of the di-RG motif (amino acids 50–70). (B) Confocal microscopy images depicting localization of specified RPS17 amino acids attached to triple eYFP protein. Arrows denote the same single nucleoli in each set of panels. Scale bars on merged images represent 10 μm.

Mentions: Truncation mutations that bestowed nuclear import functionality to triple eYFP produced a diffuse nuclear fluorescence (Fig 3B panels b, f, and h) and diffuse nuclear fluorescence with a reduced quantity of nucleolar localization (Fig 3B panel e) while the full-length RPS17 protein was strongly nucleolar during steady state conditions (Fig 2B bottom). This suggested that determinants other than single nuclear localization signals were required to retain the RPS17 protein within nucleoli. Therefore, we created constructs in which either amino acids 1–13 (NLS1) or amino acids 32–50 (NLS2) in the presence or absence of amino acids 60–70 (di-RG motif) to test whether two nuclear localization signals in tandem or if a single nuclear localization signal along with a di-RG motif [35] was sufficient for nucleolar trafficking (Fig 6A). Expressing amino acids 1–13 and 32–50 in tandem with triple eYFP resulted in diffuse nuclear fluorescence that excluded nucleoli (Fig 6B panel a). We further added amino acids 60–70, containing the di-RG motif, along with either NLS1 or NLS2. Interestingly, amino acids 1–13 in tandem with amino acids 60–70 resulted in both diffuse nuclear and nucleolar localization (Fig 6B panel b), whereas amino acids 32–50 in tandem with 60–70 produced diffuse nuclear localization with occasional nucleolar localization (Fig 6B panels c and d). Expressing amino acids 1–13, 32–50, and 60–70 in tandem resulted in different phenotypes including diffuse nuclear, punctate nuclear, and nucleolar staining (Fig 6B panels e and f). Expression of the RG domain alone remained predominantly cytoplasmic as expected since it did not contain a nuclear localization signal (Fig 6B panel g).


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

Kenney SP, Meng XJ - PLoS ONE (2015)

Mapping the determinants of nucleolar localization within RPS17.(A) Schematic depicting amino acids from nuclear localization sequence (NLS) 1 (amino acids 1–13) and NLS 2 (amino acids 32–50) in concert with each other separated via a glycine serine flexible linker or individually in the presence of the di-RG motif (amino acids 50–70). (B) Confocal microscopy images depicting localization of specified RPS17 amino acids attached to triple eYFP protein. Arrows denote the same single nucleoli in each set of panels. 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.g006: Mapping the determinants of nucleolar localization within RPS17.(A) Schematic depicting amino acids from nuclear localization sequence (NLS) 1 (amino acids 1–13) and NLS 2 (amino acids 32–50) in concert with each other separated via a glycine serine flexible linker or individually in the presence of the di-RG motif (amino acids 50–70). (B) Confocal microscopy images depicting localization of specified RPS17 amino acids attached to triple eYFP protein. Arrows denote the same single nucleoli in each set of panels. Scale bars on merged images represent 10 μm.
Mentions: Truncation mutations that bestowed nuclear import functionality to triple eYFP produced a diffuse nuclear fluorescence (Fig 3B panels b, f, and h) and diffuse nuclear fluorescence with a reduced quantity of nucleolar localization (Fig 3B panel e) while the full-length RPS17 protein was strongly nucleolar during steady state conditions (Fig 2B bottom). This suggested that determinants other than single nuclear localization signals were required to retain the RPS17 protein within nucleoli. Therefore, we created constructs in which either amino acids 1–13 (NLS1) or amino acids 32–50 (NLS2) in the presence or absence of amino acids 60–70 (di-RG motif) to test whether two nuclear localization signals in tandem or if a single nuclear localization signal along with a di-RG motif [35] was sufficient for nucleolar trafficking (Fig 6A). Expressing amino acids 1–13 and 32–50 in tandem with triple eYFP resulted in diffuse nuclear fluorescence that excluded nucleoli (Fig 6B panel a). We further added amino acids 60–70, containing the di-RG motif, along with either NLS1 or NLS2. Interestingly, amino acids 1–13 in tandem with amino acids 60–70 resulted in both diffuse nuclear and nucleolar localization (Fig 6B panel b), whereas amino acids 32–50 in tandem with 60–70 produced diffuse nuclear localization with occasional nucleolar localization (Fig 6B panels c and d). Expressing amino acids 1–13, 32–50, and 60–70 in tandem resulted in different phenotypes including diffuse nuclear, punctate nuclear, and nucleolar staining (Fig 6B panels e and f). Expression of the RG domain alone remained predominantly cytoplasmic as expected since it did not contain a nuclear localization signal (Fig 6B panel g).

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