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Insights into the origin of the nuclear localization signals in conserved ribosomal proteins.

Melnikov S, Ben-Shem A, Yusupova G, Yusupov M - Nat Commun (2015)

Bottom Line: Eukaryotic ribosomal proteins, unlike their bacterial homologues, possess nuclear localization signals (NLSs) to enter the cell nucleus during ribosome assembly.Here we provide a comprehensive comparison of bacterial and eukaryotic ribosomes to show that NLSs appear in conserved ribosomal proteins via remodelling of their RNA-binding domains.This finding enabled us to identify previously unknown NLSs in ribosomal proteins from humans, and suggests that, apart from promoting protein transport, NLSs may facilitate folding of ribosomal RNA.

View Article: PubMed Central - PubMed

Affiliation: 1] Strasbourg University, 4 Rue Blaise Pascal, 67081 Strasbourg, France [2] Institute of Genetics and Molecular and Cellular Biology, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France [3] Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06511, USA.

ABSTRACT
Eukaryotic ribosomal proteins, unlike their bacterial homologues, possess nuclear localization signals (NLSs) to enter the cell nucleus during ribosome assembly. Here we provide a comprehensive comparison of bacterial and eukaryotic ribosomes to show that NLSs appear in conserved ribosomal proteins via remodelling of their RNA-binding domains. This finding enabled us to identify previously unknown NLSs in ribosomal proteins from humans, and suggests that, apart from promoting protein transport, NLSs may facilitate folding of ribosomal RNA.

No MeSH data available.


Related in: MedlinePlus

Different structure of homologous ribosomal proteins within highly conserved rRNA pockets may point to the location of unknown nuclear localization signals.Exemplified by identification of NLS in human ribosomal protein uS12. (a) Structure of protein uS12, coloured according to structural conservation: conserved fold is shown in grey and bacteria- and eukaryote-specific in blue and red, respectively; surrounding rRNA is shown schematically with labels indicating 16S/18S rRNA helices. Protein uS12 is one of the 15 ribosomal proteins in which extensions have different folds in bacteria and eukaryotes, despite being bound to nearly identical rRNA cavities of bacterial and eukaryotic ribosomes. (b–h) eGFP fluorescence (top panels) and phase-contrast (bottom panels) snapshots of human cell line HEK293, which express eGFP fusions of human or E. coli protein uS12. Arrows point to nucleoli. All scale bars represent 10 μm. For each sample, eGFP localization was examined in 200 cells. Cells, in which eGFP distribution pattern was common for >95% of the analysed population, were used for imaging. The experiments were replicated three times. (b) eGFP alone (a negative control). (c) Human uS12–eGFP fusion. (d) E. coli uS12–eGFP fusion. (e) A protein hybrid carrying the N terminus of human uS12 and the globular domain of E. coli uS12. (f) A protein hybrid carrying the N terminus of E. coli uS12 and the globular domain of human uS12. (b–h) Identifying NLSs within human uS12 shows that its N-terminal extension (N-H. sapiens) carries NLS activity – by contrast to analogous extension in bacterial uS12 (N-E. coli).
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f2: Different structure of homologous ribosomal proteins within highly conserved rRNA pockets may point to the location of unknown nuclear localization signals.Exemplified by identification of NLS in human ribosomal protein uS12. (a) Structure of protein uS12, coloured according to structural conservation: conserved fold is shown in grey and bacteria- and eukaryote-specific in blue and red, respectively; surrounding rRNA is shown schematically with labels indicating 16S/18S rRNA helices. Protein uS12 is one of the 15 ribosomal proteins in which extensions have different folds in bacteria and eukaryotes, despite being bound to nearly identical rRNA cavities of bacterial and eukaryotic ribosomes. (b–h) eGFP fluorescence (top panels) and phase-contrast (bottom panels) snapshots of human cell line HEK293, which express eGFP fusions of human or E. coli protein uS12. Arrows point to nucleoli. All scale bars represent 10 μm. For each sample, eGFP localization was examined in 200 cells. Cells, in which eGFP distribution pattern was common for >95% of the analysed population, were used for imaging. The experiments were replicated three times. (b) eGFP alone (a negative control). (c) Human uS12–eGFP fusion. (d) E. coli uS12–eGFP fusion. (e) A protein hybrid carrying the N terminus of human uS12 and the globular domain of E. coli uS12. (f) A protein hybrid carrying the N terminus of E. coli uS12 and the globular domain of human uS12. (b–h) Identifying NLSs within human uS12 shows that its N-terminal extension (N-H. sapiens) carries NLS activity – by contrast to analogous extension in bacterial uS12 (N-E. coli).

Mentions: Having elucidated previously unknown consensus structural features of NLSs of ribosomal proteins, we next endeavoured to use our structural observations to uncover unknown NLSs. For this purpose, we examined how many homologous proteins have different folds of their rRNA-binding domains within similar rRNA cavities of bacterial and eukaryotic ribosomes. We preformed structural comparison of all homologous proteins from eukaryotic (S. cerevisiae) and bacterial (E. coli) ribosomes and, where necessary, analysed surrounding of ribosomal proteins in the ribosome structure (Supplementary Online Methods). This approach allowed us to resolve ambiguity between previous sequence1516 and secondary structure alignments14 and correct numerous mistakes in the correspondence between the residues of uS3, uS4, uS11, uS12, uS13, uS15, uS19, uL2, uL3, uL4, uL5, uL9, uL13, uL15 and uL18 (Supplementary Fig. 2). In total, we found that 27 out of 32 conserved proteins possess differently folded segments within their rRNA-binding domains between bacteria and eukaryotes, despite overall conserved structure of the adjacent rRNA: in fifteen proteins, seemingly conserved extensions have different fold in bacteria and eukaryotes, and, in seventeen proteins, extensions differ in size despite the high conservation of the surrounding rRNA (Supplementary Fig. 2). To verify if these structural differences could indeed point to the location of the NLSs, we selected proteins uS12 and uL24, whose rRNA-binding domains are among the most divergent in the small and the large ribosomal subunits, respectively (Fig. 2a, Supplementary Fig. 3). Next, we expressed uS12 fused to enhanced green fluorescent protein (eGFP) in human cells. Unlike a control eGFP sample (Fig. 2b), human uS12–eGFP fusion accumulated in the nucleoli, indicating the presence of NLS (Fig. 2c, Supplementary Fig. 4a). By contrast, the E. coli uS12–eGFP fusion was distributed between the nucleus and the cytoplasm and largely excluded from the nucleoli, consistent with the absence of NLSs in bacteria (Fig. 2d, Supplementary Fig. 4b). Next, we replaced the N terminus of E. coli uS12 with the N terminus of human uS12, a structure that is highly divergent between the two organisms despite high conservation of rRNA (Fig. 2a). We found that the resulting chimeric protein gained nucleolar accumulation in human cell lines HEK293 (Fig. 2e, Supplementary Fig. 4c). Subsequently, when the extension of human uS12 was replaced by the N terminus of its bacterial homologue, the resulting protein was no longer able to accumulate in the nucleolus (Fig. 2f, Supplementary Fig. 4d). Finally, upon examining isolated individual domains we showed that the N terminus of human uS12 alone is sufficient for the nucleolar accumulation of eGFP (Fig. 2g), whereas the globular domain alone is insufficient (Fig. 2h), indicating that the N-terminal segment indeed carries the NLS activity. Similarly, we identified the NLSs of ribosomal protein uL24, residing within structurally diverged N- and C-termini of this protein (Supplementary Fig. 3).


Insights into the origin of the nuclear localization signals in conserved ribosomal proteins.

Melnikov S, Ben-Shem A, Yusupova G, Yusupov M - Nat Commun (2015)

Different structure of homologous ribosomal proteins within highly conserved rRNA pockets may point to the location of unknown nuclear localization signals.Exemplified by identification of NLS in human ribosomal protein uS12. (a) Structure of protein uS12, coloured according to structural conservation: conserved fold is shown in grey and bacteria- and eukaryote-specific in blue and red, respectively; surrounding rRNA is shown schematically with labels indicating 16S/18S rRNA helices. Protein uS12 is one of the 15 ribosomal proteins in which extensions have different folds in bacteria and eukaryotes, despite being bound to nearly identical rRNA cavities of bacterial and eukaryotic ribosomes. (b–h) eGFP fluorescence (top panels) and phase-contrast (bottom panels) snapshots of human cell line HEK293, which express eGFP fusions of human or E. coli protein uS12. Arrows point to nucleoli. All scale bars represent 10 μm. For each sample, eGFP localization was examined in 200 cells. Cells, in which eGFP distribution pattern was common for >95% of the analysed population, were used for imaging. The experiments were replicated three times. (b) eGFP alone (a negative control). (c) Human uS12–eGFP fusion. (d) E. coli uS12–eGFP fusion. (e) A protein hybrid carrying the N terminus of human uS12 and the globular domain of E. coli uS12. (f) A protein hybrid carrying the N terminus of E. coli uS12 and the globular domain of human uS12. (b–h) Identifying NLSs within human uS12 shows that its N-terminal extension (N-H. sapiens) carries NLS activity – by contrast to analogous extension in bacterial uS12 (N-E. coli).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490412&req=5

f2: Different structure of homologous ribosomal proteins within highly conserved rRNA pockets may point to the location of unknown nuclear localization signals.Exemplified by identification of NLS in human ribosomal protein uS12. (a) Structure of protein uS12, coloured according to structural conservation: conserved fold is shown in grey and bacteria- and eukaryote-specific in blue and red, respectively; surrounding rRNA is shown schematically with labels indicating 16S/18S rRNA helices. Protein uS12 is one of the 15 ribosomal proteins in which extensions have different folds in bacteria and eukaryotes, despite being bound to nearly identical rRNA cavities of bacterial and eukaryotic ribosomes. (b–h) eGFP fluorescence (top panels) and phase-contrast (bottom panels) snapshots of human cell line HEK293, which express eGFP fusions of human or E. coli protein uS12. Arrows point to nucleoli. All scale bars represent 10 μm. For each sample, eGFP localization was examined in 200 cells. Cells, in which eGFP distribution pattern was common for >95% of the analysed population, were used for imaging. The experiments were replicated three times. (b) eGFP alone (a negative control). (c) Human uS12–eGFP fusion. (d) E. coli uS12–eGFP fusion. (e) A protein hybrid carrying the N terminus of human uS12 and the globular domain of E. coli uS12. (f) A protein hybrid carrying the N terminus of E. coli uS12 and the globular domain of human uS12. (b–h) Identifying NLSs within human uS12 shows that its N-terminal extension (N-H. sapiens) carries NLS activity – by contrast to analogous extension in bacterial uS12 (N-E. coli).
Mentions: Having elucidated previously unknown consensus structural features of NLSs of ribosomal proteins, we next endeavoured to use our structural observations to uncover unknown NLSs. For this purpose, we examined how many homologous proteins have different folds of their rRNA-binding domains within similar rRNA cavities of bacterial and eukaryotic ribosomes. We preformed structural comparison of all homologous proteins from eukaryotic (S. cerevisiae) and bacterial (E. coli) ribosomes and, where necessary, analysed surrounding of ribosomal proteins in the ribosome structure (Supplementary Online Methods). This approach allowed us to resolve ambiguity between previous sequence1516 and secondary structure alignments14 and correct numerous mistakes in the correspondence between the residues of uS3, uS4, uS11, uS12, uS13, uS15, uS19, uL2, uL3, uL4, uL5, uL9, uL13, uL15 and uL18 (Supplementary Fig. 2). In total, we found that 27 out of 32 conserved proteins possess differently folded segments within their rRNA-binding domains between bacteria and eukaryotes, despite overall conserved structure of the adjacent rRNA: in fifteen proteins, seemingly conserved extensions have different fold in bacteria and eukaryotes, and, in seventeen proteins, extensions differ in size despite the high conservation of the surrounding rRNA (Supplementary Fig. 2). To verify if these structural differences could indeed point to the location of the NLSs, we selected proteins uS12 and uL24, whose rRNA-binding domains are among the most divergent in the small and the large ribosomal subunits, respectively (Fig. 2a, Supplementary Fig. 3). Next, we expressed uS12 fused to enhanced green fluorescent protein (eGFP) in human cells. Unlike a control eGFP sample (Fig. 2b), human uS12–eGFP fusion accumulated in the nucleoli, indicating the presence of NLS (Fig. 2c, Supplementary Fig. 4a). By contrast, the E. coli uS12–eGFP fusion was distributed between the nucleus and the cytoplasm and largely excluded from the nucleoli, consistent with the absence of NLSs in bacteria (Fig. 2d, Supplementary Fig. 4b). Next, we replaced the N terminus of E. coli uS12 with the N terminus of human uS12, a structure that is highly divergent between the two organisms despite high conservation of rRNA (Fig. 2a). We found that the resulting chimeric protein gained nucleolar accumulation in human cell lines HEK293 (Fig. 2e, Supplementary Fig. 4c). Subsequently, when the extension of human uS12 was replaced by the N terminus of its bacterial homologue, the resulting protein was no longer able to accumulate in the nucleolus (Fig. 2f, Supplementary Fig. 4d). Finally, upon examining isolated individual domains we showed that the N terminus of human uS12 alone is sufficient for the nucleolar accumulation of eGFP (Fig. 2g), whereas the globular domain alone is insufficient (Fig. 2h), indicating that the N-terminal segment indeed carries the NLS activity. Similarly, we identified the NLSs of ribosomal protein uL24, residing within structurally diverged N- and C-termini of this protein (Supplementary Fig. 3).

Bottom Line: Eukaryotic ribosomal proteins, unlike their bacterial homologues, possess nuclear localization signals (NLSs) to enter the cell nucleus during ribosome assembly.Here we provide a comprehensive comparison of bacterial and eukaryotic ribosomes to show that NLSs appear in conserved ribosomal proteins via remodelling of their RNA-binding domains.This finding enabled us to identify previously unknown NLSs in ribosomal proteins from humans, and suggests that, apart from promoting protein transport, NLSs may facilitate folding of ribosomal RNA.

View Article: PubMed Central - PubMed

Affiliation: 1] Strasbourg University, 4 Rue Blaise Pascal, 67081 Strasbourg, France [2] Institute of Genetics and Molecular and Cellular Biology, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France [3] Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06511, USA.

ABSTRACT
Eukaryotic ribosomal proteins, unlike their bacterial homologues, possess nuclear localization signals (NLSs) to enter the cell nucleus during ribosome assembly. Here we provide a comprehensive comparison of bacterial and eukaryotic ribosomes to show that NLSs appear in conserved ribosomal proteins via remodelling of their RNA-binding domains. This finding enabled us to identify previously unknown NLSs in ribosomal proteins from humans, and suggests that, apart from promoting protein transport, NLSs may facilitate folding of ribosomal RNA.

No MeSH data available.


Related in: MedlinePlus