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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

Subcellular localization of RPS17 attached to triple yellow fluorescent protein (eYFP).(A) Schematic depiction of RPS17 as a carboxy-terminal fusion to RPS17. (B) RPS17 was capable of trafficking triple YFP to punctate spots within the nucleus as observed via confocal microscopy. Triple eYFP alone was predominantly cytoplasmic (Top) whereas RPS17 triple eYFP was in punctate spots within the nucleus (Bottom). (C) RPS17 triple eYFP is located within nucleoli. RPS17 triple eYFP colocalized with fibrillarin CFP (nucleolar marker) but not promyelocytic leukemia (PML) protein (PML body marker). Scale bars on merged images represent 10 μm.
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pone.0124396.g002: Subcellular localization of RPS17 attached to triple yellow fluorescent protein (eYFP).(A) Schematic depiction of RPS17 as a carboxy-terminal fusion to RPS17. (B) RPS17 was capable of trafficking triple YFP to punctate spots within the nucleus as observed via confocal microscopy. Triple eYFP alone was predominantly cytoplasmic (Top) whereas RPS17 triple eYFP was in punctate spots within the nucleus (Bottom). (C) RPS17 triple eYFP is located within nucleoli. RPS17 triple eYFP colocalized with fibrillarin CFP (nucleolar marker) but not promyelocytic leukemia (PML) protein (PML body marker). Scale bars on merged images represent 10 μm.

Mentions: Based on our NLS predictions from NLS mapper and ELM analysis in conjunction with the detection of endogenous RPS17 expression in the nucleus, it appeared that RPS17 had an active nuclear localization signal. To experimentally demonstrate the existence of active nuclear import signals, we cloned specific fragments of the RPS17 coding sequence in frame with three consecutive copies of enhanced yellow fluorescent protein (eYFP)(Fig 2A). For triple eYFP protein to be found in the nucleus, the amino acids attached to it must contain an active nuclear localization signal. As expected, when transiently expressed in Huh7 liver cells, triple YFP with no additional amino acid sequences localized predominantly to the cytoplasm (Fig 2B top). Full-length RPS17 protein attached to triple eYFP localized to distinct regions within the nucleus both large puncta resembling nucleoli and many smaller puncta that only occasionally appeared to coincide with nucleoli (Fig 2B bottom, data not shown). Subnuclear RPS17 eYFP colocalized with fibrillarin CFP [34], a nucleolar marker, but not promyelocytic leukemia (PML) CFP, a PML body marker (Fig 2C). This data suggests that RPS17 localizes to nucleoli.


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

Kenney SP, Meng XJ - PLoS ONE (2015)

Subcellular localization of RPS17 attached to triple yellow fluorescent protein (eYFP).(A) Schematic depiction of RPS17 as a carboxy-terminal fusion to RPS17. (B) RPS17 was capable of trafficking triple YFP to punctate spots within the nucleus as observed via confocal microscopy. Triple eYFP alone was predominantly cytoplasmic (Top) whereas RPS17 triple eYFP was in punctate spots within the nucleus (Bottom). (C) RPS17 triple eYFP is located within nucleoli. RPS17 triple eYFP colocalized with fibrillarin CFP (nucleolar marker) but not promyelocytic leukemia (PML) protein (PML body marker). Scale bars on merged images represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390217&req=5

pone.0124396.g002: Subcellular localization of RPS17 attached to triple yellow fluorescent protein (eYFP).(A) Schematic depiction of RPS17 as a carboxy-terminal fusion to RPS17. (B) RPS17 was capable of trafficking triple YFP to punctate spots within the nucleus as observed via confocal microscopy. Triple eYFP alone was predominantly cytoplasmic (Top) whereas RPS17 triple eYFP was in punctate spots within the nucleus (Bottom). (C) RPS17 triple eYFP is located within nucleoli. RPS17 triple eYFP colocalized with fibrillarin CFP (nucleolar marker) but not promyelocytic leukemia (PML) protein (PML body marker). Scale bars on merged images represent 10 μm.
Mentions: Based on our NLS predictions from NLS mapper and ELM analysis in conjunction with the detection of endogenous RPS17 expression in the nucleus, it appeared that RPS17 had an active nuclear localization signal. To experimentally demonstrate the existence of active nuclear import signals, we cloned specific fragments of the RPS17 coding sequence in frame with three consecutive copies of enhanced yellow fluorescent protein (eYFP)(Fig 2A). For triple eYFP protein to be found in the nucleus, the amino acids attached to it must contain an active nuclear localization signal. As expected, when transiently expressed in Huh7 liver cells, triple YFP with no additional amino acid sequences localized predominantly to the cytoplasm (Fig 2B top). Full-length RPS17 protein attached to triple eYFP localized to distinct regions within the nucleus both large puncta resembling nucleoli and many smaller puncta that only occasionally appeared to coincide with nucleoli (Fig 2B bottom, data not shown). Subnuclear RPS17 eYFP colocalized with fibrillarin CFP [34], a nucleolar marker, but not promyelocytic leukemia (PML) CFP, a PML body marker (Fig 2C). This data suggests that RPS17 localizes to nucleoli.

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