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The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA.

Meyer B, Wurm JP, Kötter P, Leisegang MS, Schilling V, Buchhaupt M, Held M, Bahr U, Karas M, Heckel A, Bohnsack MT, Wöhnert J, Entian KD - Nucleic Acids Res. (2010)

Bottom Line: This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor.Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation.Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro.

View Article: PubMed Central - PubMed

Affiliation: Cluster of Excellence Frankfurt: Macromolecular Complexes, Max-von-Laue Str. 9, D-60438 Frankfurt/M., Germany.

ABSTRACT
The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.

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Dimerization and RNA-binding affinity of BCS-mutated yeast ScNep1D90G. (A) Apparent molecular weights of ScNep1 (squares) and ScNep1D90G (circles) calculated from retention volumes after gel filtrations at nine different protein concentrations (1–216 µM) are plotted versus the ScNep1 concentration to calculate the apparent monomer/dimer dissociation constants. Dissociation constants derived from the fits were 134.5 (±17) µM for wild-type ScNep1 and 5.8 (±3.2) µM for ScNep1D90G. The calculated apparent molecular weights of the monomers were 31.1 (±0.4) kDa for wild-type ScNep1 and 30.7 (±1.4) kDa for ScNep1D90G. (B) ScNep1 binding to a 5′-fluorescein labelled RNA (5′-Fl-GACUCAACACG-3′) determined by fluorescence anisotropy. Shown are mean and standard deviation of three measurements. The binding curves were fitted to a one site binding model with one binding site per monomer. Dissociation constants derived from the fit are 6.29 (±0.98) µM for wild-type ScNep1 and 0.38 (±0.04) µM for ScNep1D90G.
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Figure 5: Dimerization and RNA-binding affinity of BCS-mutated yeast ScNep1D90G. (A) Apparent molecular weights of ScNep1 (squares) and ScNep1D90G (circles) calculated from retention volumes after gel filtrations at nine different protein concentrations (1–216 µM) are plotted versus the ScNep1 concentration to calculate the apparent monomer/dimer dissociation constants. Dissociation constants derived from the fits were 134.5 (±17) µM for wild-type ScNep1 and 5.8 (±3.2) µM for ScNep1D90G. The calculated apparent molecular weights of the monomers were 31.1 (±0.4) kDa for wild-type ScNep1 and 30.7 (±1.4) kDa for ScNep1D90G. (B) ScNep1 binding to a 5′-fluorescein labelled RNA (5′-Fl-GACUCAACACG-3′) determined by fluorescence anisotropy. Shown are mean and standard deviation of three measurements. The binding curves were fitted to a one site binding model with one binding site per monomer. Dissociation constants derived from the fit are 6.29 (±0.98) µM for wild-type ScNep1 and 0.38 (±0.04) µM for ScNep1D90G.

Mentions: Analysis of the human HsNep1D86G protein showed a strongly increased interaction of the monomers in the yeast two-hybrid system (27). To biochemically confirm an enhanced BCS protein interaction yeast wild-type and yeast ScNep1D90G proteins were expressed with an N-terminal hexahistidine tag (H6) in E. coli, affinity purified and separated by gel filtration. Analytical gel filtrations over a wide range of different protein concentrations showed a concentration-dependent monomer/dimer equilibrium for the wild-type H6-ScNep1 protein (Figure S4A). At low protein concentration (1 µM) wild-type H6-ScNep1 was eluted at ∼32 kDa, which is close to the molecular mass of the monomer (calculated molecular mass, 28.9 kDa), whereas at higher H6-ScNep1 protein concentrations the equilibrium was shifted towards the dimeric form (48 kDa at protein concentration of 216 µM, calculated molecular mass: 57.8 kDa). By contrast, comparable gel filtrations with the H6-Nep1D90G protein (Figure S4B) always provided higher molecular weights as compared to the H6-ScNep1 wild-type protein. The strong difference between the H6-ScNep1 and the H6-ScNep1D90G mutant protein became even more obvious when the molecular weights were plotted against the protein concentrations (Figure 5A). These data clearly show that the ScNep1D90G mutation caused strongly enhanced dimerization as compared to the wild-type ScNep1 protein. Based on the equation for monomer/dimer equilibria the data were fitted to the curve to calculate apparent monomer/dimer dissociation constants (see ‘Materials and Methods’ section). The ScNep1D90G mutant apparent KD (5.8 ± 3.2 µM) was more than 23-fold lower as compared to the ScNep1 wild-type apparent KD (134.5 ± 17 µM), which explains the strongly enhanced dimerization of the ScNep1D90G mutant protein. The apparent molecular weights for the wild-type monomer (MWap = 31.1 kDa) and the mutant monomer (MWap = 30.7 kDa) derived from this fittings were remarkably close to the expected molecular weight of the monomer (MW = 28.9 kDa) and confirm the accuracy of the measurements.Figure 5.


The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA.

Meyer B, Wurm JP, Kötter P, Leisegang MS, Schilling V, Buchhaupt M, Held M, Bahr U, Karas M, Heckel A, Bohnsack MT, Wöhnert J, Entian KD - Nucleic Acids Res. (2010)

Dimerization and RNA-binding affinity of BCS-mutated yeast ScNep1D90G. (A) Apparent molecular weights of ScNep1 (squares) and ScNep1D90G (circles) calculated from retention volumes after gel filtrations at nine different protein concentrations (1–216 µM) are plotted versus the ScNep1 concentration to calculate the apparent monomer/dimer dissociation constants. Dissociation constants derived from the fits were 134.5 (±17) µM for wild-type ScNep1 and 5.8 (±3.2) µM for ScNep1D90G. The calculated apparent molecular weights of the monomers were 31.1 (±0.4) kDa for wild-type ScNep1 and 30.7 (±1.4) kDa for ScNep1D90G. (B) ScNep1 binding to a 5′-fluorescein labelled RNA (5′-Fl-GACUCAACACG-3′) determined by fluorescence anisotropy. Shown are mean and standard deviation of three measurements. The binding curves were fitted to a one site binding model with one binding site per monomer. Dissociation constants derived from the fit are 6.29 (±0.98) µM for wild-type ScNep1 and 0.38 (±0.04) µM for ScNep1D90G.
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Related In: Results  -  Collection

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Figure 5: Dimerization and RNA-binding affinity of BCS-mutated yeast ScNep1D90G. (A) Apparent molecular weights of ScNep1 (squares) and ScNep1D90G (circles) calculated from retention volumes after gel filtrations at nine different protein concentrations (1–216 µM) are plotted versus the ScNep1 concentration to calculate the apparent monomer/dimer dissociation constants. Dissociation constants derived from the fits were 134.5 (±17) µM for wild-type ScNep1 and 5.8 (±3.2) µM for ScNep1D90G. The calculated apparent molecular weights of the monomers were 31.1 (±0.4) kDa for wild-type ScNep1 and 30.7 (±1.4) kDa for ScNep1D90G. (B) ScNep1 binding to a 5′-fluorescein labelled RNA (5′-Fl-GACUCAACACG-3′) determined by fluorescence anisotropy. Shown are mean and standard deviation of three measurements. The binding curves were fitted to a one site binding model with one binding site per monomer. Dissociation constants derived from the fit are 6.29 (±0.98) µM for wild-type ScNep1 and 0.38 (±0.04) µM for ScNep1D90G.
Mentions: Analysis of the human HsNep1D86G protein showed a strongly increased interaction of the monomers in the yeast two-hybrid system (27). To biochemically confirm an enhanced BCS protein interaction yeast wild-type and yeast ScNep1D90G proteins were expressed with an N-terminal hexahistidine tag (H6) in E. coli, affinity purified and separated by gel filtration. Analytical gel filtrations over a wide range of different protein concentrations showed a concentration-dependent monomer/dimer equilibrium for the wild-type H6-ScNep1 protein (Figure S4A). At low protein concentration (1 µM) wild-type H6-ScNep1 was eluted at ∼32 kDa, which is close to the molecular mass of the monomer (calculated molecular mass, 28.9 kDa), whereas at higher H6-ScNep1 protein concentrations the equilibrium was shifted towards the dimeric form (48 kDa at protein concentration of 216 µM, calculated molecular mass: 57.8 kDa). By contrast, comparable gel filtrations with the H6-Nep1D90G protein (Figure S4B) always provided higher molecular weights as compared to the H6-ScNep1 wild-type protein. The strong difference between the H6-ScNep1 and the H6-ScNep1D90G mutant protein became even more obvious when the molecular weights were plotted against the protein concentrations (Figure 5A). These data clearly show that the ScNep1D90G mutation caused strongly enhanced dimerization as compared to the wild-type ScNep1 protein. Based on the equation for monomer/dimer equilibria the data were fitted to the curve to calculate apparent monomer/dimer dissociation constants (see ‘Materials and Methods’ section). The ScNep1D90G mutant apparent KD (5.8 ± 3.2 µM) was more than 23-fold lower as compared to the ScNep1 wild-type apparent KD (134.5 ± 17 µM), which explains the strongly enhanced dimerization of the ScNep1D90G mutant protein. The apparent molecular weights for the wild-type monomer (MWap = 31.1 kDa) and the mutant monomer (MWap = 30.7 kDa) derived from this fittings were remarkably close to the expected molecular weight of the monomer (MW = 28.9 kDa) and confirm the accuracy of the measurements.Figure 5.

Bottom Line: This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor.Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation.Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro.

View Article: PubMed Central - PubMed

Affiliation: Cluster of Excellence Frankfurt: Macromolecular Complexes, Max-von-Laue Str. 9, D-60438 Frankfurt/M., Germany.

ABSTRACT
The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.

Show MeSH
Related in: MedlinePlus