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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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Intracellular localization of BCS-mutated Nep1 proteins. (A) Cellular localization of yeast wild-type ScNep1 (upper part) and the D90G mutant (lower part) using confocal microscopy. Both proteins were expressed as GFP-fusion together with the ScNop56-mRFP protein as a nucleolar marker. (Left) Green fluorescence (ScNep1); (middle) red fluorescence (ScNop56); (right) merge with differential interference contrast (DIC). (B) Intracellular localization of human HsNep1 (upper part) and the HsNep1D86G mutant (lower part) after 24 h induction with 200 ng/ml doxycycline. The cellular localizations of the GFP-fusion proteins were analysed by confocal microscopy. (Left) Green fluorescence; (middle) DAPI staining; (right) merge with differential interference contrast (DIC). Scale bar: 10 µm.
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Figure 4: Intracellular localization of BCS-mutated Nep1 proteins. (A) Cellular localization of yeast wild-type ScNep1 (upper part) and the D90G mutant (lower part) using confocal microscopy. Both proteins were expressed as GFP-fusion together with the ScNop56-mRFP protein as a nucleolar marker. (Left) Green fluorescence (ScNep1); (middle) red fluorescence (ScNop56); (right) merge with differential interference contrast (DIC). (B) Intracellular localization of human HsNep1 (upper part) and the HsNep1D86G mutant (lower part) after 24 h induction with 200 ng/ml doxycycline. The cellular localizations of the GFP-fusion proteins were analysed by confocal microscopy. (Left) Green fluorescence; (middle) DAPI staining; (right) merge with differential interference contrast (DIC). Scale bar: 10 µm.

Mentions: For further analysis, the cellular localization of the yeast (ScNep1D90G) and human (HsNep1D86G) BCS proteins was analysed using GFP-fusions. In yeast, the GFP-ScNep1D90G fusion complemented growth in a ScΔnep1 deletion after its multi-copy expression. Interestingly, the BCS ScNep1D90G protein lost its exclusive nuclear localization. In GFP-ScNep1 wild-type cells a strong super-imposed signal was seen with Nop56-mRFP, which was used as a nucleolar marker, whereas for the GFP-ScNep1D90G fusion the nuclear localization was strongly reduced and it showed distinct cytoplasmic staining (Figure 4A).Figure 4.


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)

Intracellular localization of BCS-mutated Nep1 proteins. (A) Cellular localization of yeast wild-type ScNep1 (upper part) and the D90G mutant (lower part) using confocal microscopy. Both proteins were expressed as GFP-fusion together with the ScNop56-mRFP protein as a nucleolar marker. (Left) Green fluorescence (ScNep1); (middle) red fluorescence (ScNop56); (right) merge with differential interference contrast (DIC). (B) Intracellular localization of human HsNep1 (upper part) and the HsNep1D86G mutant (lower part) after 24 h induction with 200 ng/ml doxycycline. The cellular localizations of the GFP-fusion proteins were analysed by confocal microscopy. (Left) Green fluorescence; (middle) DAPI staining; (right) merge with differential interference contrast (DIC). Scale bar: 10 µm.
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Figure 4: Intracellular localization of BCS-mutated Nep1 proteins. (A) Cellular localization of yeast wild-type ScNep1 (upper part) and the D90G mutant (lower part) using confocal microscopy. Both proteins were expressed as GFP-fusion together with the ScNop56-mRFP protein as a nucleolar marker. (Left) Green fluorescence (ScNep1); (middle) red fluorescence (ScNop56); (right) merge with differential interference contrast (DIC). (B) Intracellular localization of human HsNep1 (upper part) and the HsNep1D86G mutant (lower part) after 24 h induction with 200 ng/ml doxycycline. The cellular localizations of the GFP-fusion proteins were analysed by confocal microscopy. (Left) Green fluorescence; (middle) DAPI staining; (right) merge with differential interference contrast (DIC). Scale bar: 10 µm.
Mentions: For further analysis, the cellular localization of the yeast (ScNep1D90G) and human (HsNep1D86G) BCS proteins was analysed using GFP-fusions. In yeast, the GFP-ScNep1D90G fusion complemented growth in a ScΔnep1 deletion after its multi-copy expression. Interestingly, the BCS ScNep1D90G protein lost its exclusive nuclear localization. In GFP-ScNep1 wild-type cells a strong super-imposed signal was seen with Nop56-mRFP, which was used as a nucleolar marker, whereas for the GFP-ScNep1D90G fusion the nuclear localization was strongly reduced and it showed distinct cytoplasmic staining (Figure 4A).Figure 4.

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