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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

Alignment of RPS17 proteins from Eukaryotes and Archaea.Six RPS17 proteins from Eukaryotic organisms and six RPS17 proteins from Archae organisms were aligned using ClustalW. Organisms used were Methanothermobacter thermautotrophicum (O26894), Methanococcus jannaschii (P54026), Pyrococcus abyssi (Q9V0G0), Pyrococcus horikoshii (P58503), Pyrococcus furiosus (Q8U0U1), Saccharaomyces cerevisiae (P14127), Neurospora crassa (P27770), Drosphila melanogaster (P17704), Arabidopsis thaliana RPS17C (Q9SQZ1), Canis Lupus familiaris (P63273), and Homo sapiens (P08708). Absolutely conserved amino acids are boxed. Secondary structure of the protein is denoted below the alignment based on the solution structure of M. thermoautotrophicum [47]. Regions determined to be important for nuclear localization are shown above the alignments. Figure based on sequences originally analyzed by [47].
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pone.0124396.g007: Alignment of RPS17 proteins from Eukaryotes and Archaea.Six RPS17 proteins from Eukaryotic organisms and six RPS17 proteins from Archae organisms were aligned using ClustalW. Organisms used were Methanothermobacter thermautotrophicum (O26894), Methanococcus jannaschii (P54026), Pyrococcus abyssi (Q9V0G0), Pyrococcus horikoshii (P58503), Pyrococcus furiosus (Q8U0U1), Saccharaomyces cerevisiae (P14127), Neurospora crassa (P27770), Drosphila melanogaster (P17704), Arabidopsis thaliana RPS17C (Q9SQZ1), Canis Lupus familiaris (P63273), and Homo sapiens (P08708). Absolutely conserved amino acids are boxed. Secondary structure of the protein is denoted below the alignment based on the solution structure of M. thermoautotrophicum [47]. Regions determined to be important for nuclear localization are shown above the alignments. Figure based on sequences originally analyzed by [47].

Mentions: RPS17 contains a single di-RG motif within amino acids 60–70, and at least one RG motif was found in all of the eukaryotic RPS17 proteins we examined in this study (Fig 7). Di-RG motifs are quite common (found in >1700 proteins) and known to be involved in both protein-protein and protein-RNA interactions [35]. A significant number of nucleolar localized proteins contain this di-RG motif including serine arginine-rich splicing factors (NCBI 15055543), splicing factor 3B subunit 4 (NCBI 5032069), nucleolar protein 9 (NCBI 28212272), nucleolar RNA helicases (NCBI 379317177), nucleolar and coiled-body phosphoprotein 1 (NCBI 148596949), among others, suggesting that the RPS17 protein could be using a conserved mechanism for nucleolar localization. It is interesting to note that, in species where the first RG motif in RPS17 is not absolutely conserved, a charged amino acid remains in the R position (histidine for D. melanogaster and lysine for A. thaliana and S. cerevisiae) and an amino acid with a small side chain remains in the G position (serine in D. melanogaster). This could be indicative of a mechanism requiring a charge based interaction with little steric hindrance from the neighboring amino acid side chains.


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

Kenney SP, Meng XJ - PLoS ONE (2015)

Alignment of RPS17 proteins from Eukaryotes and Archaea.Six RPS17 proteins from Eukaryotic organisms and six RPS17 proteins from Archae organisms were aligned using ClustalW. Organisms used were Methanothermobacter thermautotrophicum (O26894), Methanococcus jannaschii (P54026), Pyrococcus abyssi (Q9V0G0), Pyrococcus horikoshii (P58503), Pyrococcus furiosus (Q8U0U1), Saccharaomyces cerevisiae (P14127), Neurospora crassa (P27770), Drosphila melanogaster (P17704), Arabidopsis thaliana RPS17C (Q9SQZ1), Canis Lupus familiaris (P63273), and Homo sapiens (P08708). Absolutely conserved amino acids are boxed. Secondary structure of the protein is denoted below the alignment based on the solution structure of M. thermoautotrophicum [47]. Regions determined to be important for nuclear localization are shown above the alignments. Figure based on sequences originally analyzed by [47].
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124396.g007: Alignment of RPS17 proteins from Eukaryotes and Archaea.Six RPS17 proteins from Eukaryotic organisms and six RPS17 proteins from Archae organisms were aligned using ClustalW. Organisms used were Methanothermobacter thermautotrophicum (O26894), Methanococcus jannaschii (P54026), Pyrococcus abyssi (Q9V0G0), Pyrococcus horikoshii (P58503), Pyrococcus furiosus (Q8U0U1), Saccharaomyces cerevisiae (P14127), Neurospora crassa (P27770), Drosphila melanogaster (P17704), Arabidopsis thaliana RPS17C (Q9SQZ1), Canis Lupus familiaris (P63273), and Homo sapiens (P08708). Absolutely conserved amino acids are boxed. Secondary structure of the protein is denoted below the alignment based on the solution structure of M. thermoautotrophicum [47]. Regions determined to be important for nuclear localization are shown above the alignments. Figure based on sequences originally analyzed by [47].
Mentions: RPS17 contains a single di-RG motif within amino acids 60–70, and at least one RG motif was found in all of the eukaryotic RPS17 proteins we examined in this study (Fig 7). Di-RG motifs are quite common (found in >1700 proteins) and known to be involved in both protein-protein and protein-RNA interactions [35]. A significant number of nucleolar localized proteins contain this di-RG motif including serine arginine-rich splicing factors (NCBI 15055543), splicing factor 3B subunit 4 (NCBI 5032069), nucleolar protein 9 (NCBI 28212272), nucleolar RNA helicases (NCBI 379317177), nucleolar and coiled-body phosphoprotein 1 (NCBI 148596949), among others, suggesting that the RPS17 protein could be using a conserved mechanism for nucleolar localization. It is interesting to note that, in species where the first RG motif in RPS17 is not absolutely conserved, a charged amino acid remains in the R position (histidine for D. melanogaster and lysine for A. thaliana and S. cerevisiae) and an amino acid with a small side chain remains in the G position (serine in D. melanogaster). This could be indicative of a mechanism requiring a charge based interaction with little steric hindrance from the neighboring amino acid side chains.

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