Limits...
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.

Show MeSH

Related in: MedlinePlus

Analysis of the U1191 yeast 18S rRNA hypermodification. (A) The predicted secondary structure of a region of 18S rRNA (www.rna.ccbb.utexas.edu/) containing helix 35 and the hypermodified nucleotide m1acp3Ψ (1191 in S. cerevisiae, 1248 in H. sapiens) is shown. (B) Specific 14C-aminocarboxypropyl-labeling of yeast 18S rRNA nucleotide U1191 with L-[1-14C]-methionine. The asterisk indicates the 14C-label (top). Total RNA was isolated from ScΔmet13 cells after growth with L-[1-14C]-methionine and separated by acrylamide gel electrophoresis (bottom). (Upper part) Ethidium bromide staining of RNA gels. (Lower part) Autoradiography of RNA gels. Samples of 5 and 10 µl total RNA from wild type (CEN.NM1-4D: WT, lanes 1, 2 and 9), rDNA deleted strains with plasmid encoded wild-type rDNA (CEN.BM146-1C: Ψ1191, lanes 3 and 4), plasmid encoded rDNA mutations U1191C (CEN.BM147-1C: Ψ1191C, lanes 5 and 6), U1191A (CEN.BM148-1C: Ψ1191A, lanes 7 and 8), a ScΔsnr35 deleted strain (CEN.BM141-7G: Δsnr35, lanes 10 and 11) and a ScΔnep1 Δnop6 deleted strain (CEN.BM140-11B: Δnep1 Δnop6, lanes 12 and 13) were loaded on the gel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045603&req=5

Figure 1: Analysis of the U1191 yeast 18S rRNA hypermodification. (A) The predicted secondary structure of a region of 18S rRNA (www.rna.ccbb.utexas.edu/) containing helix 35 and the hypermodified nucleotide m1acp3Ψ (1191 in S. cerevisiae, 1248 in H. sapiens) is shown. (B) Specific 14C-aminocarboxypropyl-labeling of yeast 18S rRNA nucleotide U1191 with L-[1-14C]-methionine. The asterisk indicates the 14C-label (top). Total RNA was isolated from ScΔmet13 cells after growth with L-[1-14C]-methionine and separated by acrylamide gel electrophoresis (bottom). (Upper part) Ethidium bromide staining of RNA gels. (Lower part) Autoradiography of RNA gels. Samples of 5 and 10 µl total RNA from wild type (CEN.NM1-4D: WT, lanes 1, 2 and 9), rDNA deleted strains with plasmid encoded wild-type rDNA (CEN.BM146-1C: Ψ1191, lanes 3 and 4), plasmid encoded rDNA mutations U1191C (CEN.BM147-1C: Ψ1191C, lanes 5 and 6), U1191A (CEN.BM148-1C: Ψ1191A, lanes 7 and 8), a ScΔsnr35 deleted strain (CEN.BM141-7G: Δsnr35, lanes 10 and 11) and a ScΔnep1 Δnop6 deleted strain (CEN.BM140-11B: Δnep1 Δnop6, lanes 12 and 13) were loaded on the gel.

Mentions: As our in vitro experiments showed a specific pseudouridine N1-methyltransferase activity of MjNep1 if this Ψ is located at a position corresponding to Ψ1191 in the putative Nep1 binding site (34), we focused our attention to the region near helix 35 which contains the hypermodified nucleotide m1acp3Ψ at position 1191 (Figure 1A).Figure 1.


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)

Analysis of the U1191 yeast 18S rRNA hypermodification. (A) The predicted secondary structure of a region of 18S rRNA (www.rna.ccbb.utexas.edu/) containing helix 35 and the hypermodified nucleotide m1acp3Ψ (1191 in S. cerevisiae, 1248 in H. sapiens) is shown. (B) Specific 14C-aminocarboxypropyl-labeling of yeast 18S rRNA nucleotide U1191 with L-[1-14C]-methionine. The asterisk indicates the 14C-label (top). Total RNA was isolated from ScΔmet13 cells after growth with L-[1-14C]-methionine and separated by acrylamide gel electrophoresis (bottom). (Upper part) Ethidium bromide staining of RNA gels. (Lower part) Autoradiography of RNA gels. Samples of 5 and 10 µl total RNA from wild type (CEN.NM1-4D: WT, lanes 1, 2 and 9), rDNA deleted strains with plasmid encoded wild-type rDNA (CEN.BM146-1C: Ψ1191, lanes 3 and 4), plasmid encoded rDNA mutations U1191C (CEN.BM147-1C: Ψ1191C, lanes 5 and 6), U1191A (CEN.BM148-1C: Ψ1191A, lanes 7 and 8), a ScΔsnr35 deleted strain (CEN.BM141-7G: Δsnr35, lanes 10 and 11) and a ScΔnep1 Δnop6 deleted strain (CEN.BM140-11B: Δnep1 Δnop6, lanes 12 and 13) were loaded on the gel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045603&req=5

Figure 1: Analysis of the U1191 yeast 18S rRNA hypermodification. (A) The predicted secondary structure of a region of 18S rRNA (www.rna.ccbb.utexas.edu/) containing helix 35 and the hypermodified nucleotide m1acp3Ψ (1191 in S. cerevisiae, 1248 in H. sapiens) is shown. (B) Specific 14C-aminocarboxypropyl-labeling of yeast 18S rRNA nucleotide U1191 with L-[1-14C]-methionine. The asterisk indicates the 14C-label (top). Total RNA was isolated from ScΔmet13 cells after growth with L-[1-14C]-methionine and separated by acrylamide gel electrophoresis (bottom). (Upper part) Ethidium bromide staining of RNA gels. (Lower part) Autoradiography of RNA gels. Samples of 5 and 10 µl total RNA from wild type (CEN.NM1-4D: WT, lanes 1, 2 and 9), rDNA deleted strains with plasmid encoded wild-type rDNA (CEN.BM146-1C: Ψ1191, lanes 3 and 4), plasmid encoded rDNA mutations U1191C (CEN.BM147-1C: Ψ1191C, lanes 5 and 6), U1191A (CEN.BM148-1C: Ψ1191A, lanes 7 and 8), a ScΔsnr35 deleted strain (CEN.BM141-7G: Δsnr35, lanes 10 and 11) and a ScΔnep1 Δnop6 deleted strain (CEN.BM140-11B: Δnep1 Δnop6, lanes 12 and 13) were loaded on the gel.
Mentions: As our in vitro experiments showed a specific pseudouridine N1-methyltransferase activity of MjNep1 if this Ψ is located at a position corresponding to Ψ1191 in the putative Nep1 binding site (34), we focused our attention to the region near helix 35 which contains the hypermodified nucleotide m1acp3Ψ at position 1191 (Figure 1A).Figure 1.

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