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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 truncated RPS17 proteins containing amino acid mutations within putative nuclear localization signals.(A) Truncated RPS17 fragments that bestowed nuclear import to triple eYFP were mutated to determine the critical residues responsible for nuclear import. This schematic depicts the amino acid sequences of 1–13, 43–61, and 31–50 (capable of nuclear import) along with basic amino acids mutated to alanine or threonine. (B) Confocal microscopy images of RPS17 truncations with and without basic amino acids mutated to alanine or threonine. Lysines, 10/11, lysine and arginine 48/49, and lysines 44/45 were important for nuclear import in the context of amino acids 1–13, and 43–61 respectively, however, 44/45 was dispensable in the context of amino acids 32–50. Scale bars on merged images represent 10 μm.
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pone.0124396.g004: Localization of truncated RPS17 proteins containing amino acid mutations within putative nuclear localization signals.(A) Truncated RPS17 fragments that bestowed nuclear import to triple eYFP were mutated to determine the critical residues responsible for nuclear import. This schematic depicts the amino acid sequences of 1–13, 43–61, and 31–50 (capable of nuclear import) along with basic amino acids mutated to alanine or threonine. (B) Confocal microscopy images of RPS17 truncations with and without basic amino acids mutated to alanine or threonine. Lysines, 10/11, lysine and arginine 48/49, and lysines 44/45 were important for nuclear import in the context of amino acids 1–13, and 43–61 respectively, however, 44/45 was dispensable in the context of amino acids 32–50. Scale bars on merged images represent 10 μm.

Mentions: To further map the nuclear localization signals within RPS17, we attached several smaller stretches of amino acids from RPS17 to eYFP3. To test whether the ELM predicted monopartite NLS (amino acids 42–48) (Fig 3A) was functional, we inserted RPS17 encoding amino acids 39–51 in frame with eYFP3. RPS17 39–51 eYFP3 was localized to the cytoplasm suggesting that the predicted monopartite NLS was not sufficient for nuclear import (Fig 3 panel g). A slightly larger region of RPS17 encompassing amino acids 32–50 and 31–50 including the ELM predicted bipartite NLS (amino acids 32–48) was functional for nuclear import (Fig 3 panel h and data not shown). Surprisingly, another fragment encompassing amino acids 43–61 was also capable of importing triple eYFP into the nucleus, although it appears as though import or nuclear retention was less efficient as the protein was also diffuse within the cytoplasm (Fig 3B panel f and Fig 4B panel c).


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 truncated RPS17 proteins containing amino acid mutations within putative nuclear localization signals.(A) Truncated RPS17 fragments that bestowed nuclear import to triple eYFP were mutated to determine the critical residues responsible for nuclear import. This schematic depicts the amino acid sequences of 1–13, 43–61, and 31–50 (capable of nuclear import) along with basic amino acids mutated to alanine or threonine. (B) Confocal microscopy images of RPS17 truncations with and without basic amino acids mutated to alanine or threonine. Lysines, 10/11, lysine and arginine 48/49, and lysines 44/45 were important for nuclear import in the context of amino acids 1–13, and 43–61 respectively, however, 44/45 was dispensable in the context of amino acids 32–50. 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.g004: Localization of truncated RPS17 proteins containing amino acid mutations within putative nuclear localization signals.(A) Truncated RPS17 fragments that bestowed nuclear import to triple eYFP were mutated to determine the critical residues responsible for nuclear import. This schematic depicts the amino acid sequences of 1–13, 43–61, and 31–50 (capable of nuclear import) along with basic amino acids mutated to alanine or threonine. (B) Confocal microscopy images of RPS17 truncations with and without basic amino acids mutated to alanine or threonine. Lysines, 10/11, lysine and arginine 48/49, and lysines 44/45 were important for nuclear import in the context of amino acids 1–13, and 43–61 respectively, however, 44/45 was dispensable in the context of amino acids 32–50. Scale bars on merged images represent 10 μm.
Mentions: To further map the nuclear localization signals within RPS17, we attached several smaller stretches of amino acids from RPS17 to eYFP3. To test whether the ELM predicted monopartite NLS (amino acids 42–48) (Fig 3A) was functional, we inserted RPS17 encoding amino acids 39–51 in frame with eYFP3. RPS17 39–51 eYFP3 was localized to the cytoplasm suggesting that the predicted monopartite NLS was not sufficient for nuclear import (Fig 3 panel g). A slightly larger region of RPS17 encompassing amino acids 32–50 and 31–50 including the ELM predicted bipartite NLS (amino acids 32–48) was functional for nuclear import (Fig 3 panel h and data not shown). Surprisingly, another fragment encompassing amino acids 43–61 was also capable of importing triple eYFP into the nucleus, although it appears as though import or nuclear retention was less efficient as the protein was also diffuse within the cytoplasm (Fig 3B panel f and Fig 4B panel c).

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