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Information on EC 2.1.1.260 - rRNA small subunit pseudouridine methyltransferase Nep1
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EC Tree
2.1.1.260 rRNA small subunit pseudouridine methyltransferase Nep1IUBMB Comments
This enzyme, which occurs in both prokaryotes and eukaryotes, recognizes specific pseudouridine residues (Psi) in small subunits of ribosomal RNA based on the local RNA structure. It recognizes Psi914 in 16S rRNA from the archaeon Methanocaldococcus jannaschii, Psi1191 in yeast 18S rRNA, and Psi1248 in human 18S rRNA.
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Word Map
- 2.1.1.260
- rrnas
- jannaschii
-
methanocaldococcus
- nucleolus
-
hypermodified
- s-adenosylmethionine
- archaea
-
three-hybrid
-
rna-binding
The expected taxonomic range for this enzyme is: Archaea, Eukaryota, Bacteria
Reaction Schemes
+
=
+
+
pseudouridine1191 in yeast 18S rRNA
=
+
N1-methylpseudouridine1191 in yeast 18S rRNA
Synonyms
nep1p, essential for mitotic growth 1, nucleolar essential protein 1, n1-specific pseudouridine methyltransferase, nep1 methyltransferase, pseudouridine n1-methyltransferase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Bowen-Conradi syndrome protein
Emg1
Emg1p
essential for mitotic growth 1
N1-specific pseudouridine methyltransferase
Nep1
Nep1 methyltransferase
Nep1p
nucleolar essential protein 1
pseudouridine N1-methyltransferase
pseudouridine-N1-specific methyltransferase
Nep1
-
-
-
-
nucleolar essential protein 1
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA = S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
-
-
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:18S rRNA (pseudouridine1191-N1)-methyltransferase
This enzyme, which occurs in both prokaryotes and eukaryotes, recognizes specific pseudouridine residues (Psi) in small subunits of ribosomal RNA based on the local RNA structure. It recognizes Psi914 in 16S rRNA from the archaeon Methanocaldococcus jannaschii, Psi1191 in yeast 18S rRNA, and Psi1248 in human 18S rRNA.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-methionine binding structure, overview
S-adenosyl-L-homocysteine binding structure, overview
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
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RNA-oligonucleotides corresponding to nucleotides 1245-1255 of human 18S rRNA and containing pseudouridine (5'-GACWCAACACG-3') at position 1248. This position corresponds to nucleotide 914 of Methanocaldococcus jannaschii 16S rRNA and to nt 1191 of yeast 18S rRNA
product identification by MALDI-mass spectrometrical analysis
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
product identification by 1H-NMR-spectroscopy
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-methionine binding site structure, overview
S-adenosyl-L-homocysteine binds to Nep1 at a preformed binding site that is topologically equivalent to the cofactor binding site in other SPOUT-class methyltransferases, intermolecular interactions, overview
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-methionine binding site structure, overview
S-adenosyl-L-homocysteine binds to Nep1 at a preformed binding site that is topologically equivalent to the cofactor binding site in other SPOUT-class methyltransferases, intermolecular interactions, overview
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
Nep1 catalyzes the psi1191 methylation in vivo. 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)-pseudouridine
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-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
S-adenosyl-L-methionine binding structure, overview
S-adenosyl-L-homocysteine binding structure, overview
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
-
?
additional information
?
-
-
RNA-binding specificity by in vitro using fluorescence quenching assays and yeast three-hybrid screening. Identification of the binding site for methylation target RNAs by NMR-spectroscopy, overview
-
-
?
additional information
?
-
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the Methanocaldococcus jannaschii Nep1 binds to the Saccharomyces cerevisiae Nep1 RNA consensus sequence 5'-UUCAAC-3' in the Methanocaldococcus jannaschii 16S rRNA. The region of Methanocaldococcus jannaschii 16S rRNA is equivalent to nt 1190-1195 in yeast 18S rRNA including the m1acp3-psi nucleotide at position 1191. And binding of MjNep1 to a 6mer RNA containing the consensus sequence 5'-UUCAAC-3'. Even a 5'-truncated RNA lacking the first uridine of the consensus sequence is bound with comparable affinity. RNA-binding specificity by in vitro using fluorescence quenching assays and yeast three-hybrid screening. Identification of the binding site for methylation target RNAs by NMR-spectroscopy, overview
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?
additional information
?
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Nep1 target site identification by mass spectrometry, isotope-labeling, and fluorescence anisotropy measurements
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-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
additional information
?
-
-
Nep1 target site identification by mass spectrometry, isotope-labeling, and fluorescence anisotropy measurements
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
Nep1 catalyzes the psi1191 methylation in vivo. 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)-pseudouridine
-
-
?
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
-
-
-
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?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
sinefungin
sinefungin binds to Nep1 at a preformed binding site that is topologically equivalent to the cofactor binding site in other SPOUT-class methyltransferases
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
derived from the clinical isolate SC5314, gene NEP1
UniProt
brenda
and isogenic strains, gene YLR186w or NEP1 or EMG1
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-
brenda
derived from the clinical isolate SC5314, gene NEP1
UniProt
brenda
strains from BY strain background or isogenic to strain CEN.PK2, gene nep1
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-
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
the spindle/microtubule association is specific for ScNep1p
brenda
-
the spindle/microtubule association is specific for ScNep1p
-
brenda
-
the spindle/microtubule association is specific for ScNep1p
brenda
-
the spindle/microtubule association is specific for ScNep1p
-
brenda
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
evolution
-
Nep1 belongs to the SPOUT-class RNA methyltransferases, Nep1 subfamily
evolution
-
Nep1 belongs to the SPOUT-class RNA methyltransferases, Nep1 subfamily
evolution
-
Nep1 is a member of the SPOUT-family of methyltransferases
malfunction
-
a temperature-sensitive ScNEP1ts1 allele is isolated and reveals a strongly increased sensitivity to paromomycin, a translational inhibitor which binds to RNA, indicating that ribosome biogenesis within the nucleolus is probably affected. Candida albicans and human NEP1 heterologously complement the essential phenotype in a Saccharomyces cerevisiae nep1 deletion mutant, the ScNEP1 spindle/microtubule phenotype is not found with HsNEP1 and CaNEP1
malfunction
addition of SAM to the medium restores growth at elevated temperatures in yeast with temperature sensitive mutants of the yeast Nep1 protein
malfunction
-
lethal phenotype of a DELTAnep1 deletion, deletions in ribosome quality and functional control genes lead to DELTAnep1 growth deficiency. Except for DELTArps18b, deletions in the identified ribosome biogenesis genes are synthetically lethal with DELTAnep1. The DELTAutp30 deletion itself has no phenotype but it enforces all nep1-1ts mutant phenotypes, utp30 overexpression partially restores the nep1-1ts growth deficiency
malfunction
-
mutations in Nep1 result in decreased methyl donor binding, but do not result in lethality
malfunction
-
Nep1 mutation D86G causes the Bowen-Conradi syndrome, BCS, that results in severe pre and postnatal growth and psychomotor retardation, microcephaly, micrognathia, rocker bottom feet and early childhood death, overview. Human HsNep1D86G protein shows a strongly increased interaction of the monomers
malfunction
-
restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine also after preventing U1191 methylation in a DElTAsnr35 mutant. Nep1 methyltransferase activity is not affected upon introduction of the Bowen-Conradi syndrome, BCS, D86G mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro
malfunction
-
a temperature-sensitive ScNEP1ts1 allele is isolated and reveals a strongly increased sensitivity to paromomycin, a translational inhibitor which binds to RNA, indicating that ribosome biogenesis within the nucleolus is probably affected. Candida albicans and human NEP1 heterologously complement the essential phenotype in a Saccharomyces cerevisiae nep1 deletion mutant, the ScNEP1 spindle/microtubule phenotype is not found with HsNEP1 and CaNEP1
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malfunction
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addition of SAM to the medium restores growth at elevated temperatures in yeast with temperature sensitive mutants of the yeast Nep1 protein
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metabolism
-
Nep1 interacting genes correspond to ribosome biogenesis, i.e. RPS18A, RPS18B, RRP8, EFG1, UTP30, to ribosome quality control, i.e. UBP3, BRE5, UBP6, and to ribosome functional control, i.e. DOM34, no-go decay
metabolism
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replacement of U1191 by any other base caused significant growth deficiencies
physiological function
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MjNep1 binds its RNA target along an extended basic surface cleft at the dimer interface that involves both insertions to the SPOUT-class core fold in an orientation consistent with the proposed methylation of nt 914 in Methanocaldococcus jannaschii 16S rRNA
physiological function
Nep1 is a genuine rRNA methyltransferase and is essential for ribosome biogenesis
physiological function
Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine, it is required for 18S rRNA maturation. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis, structure-function relationship, overview
physiological function
-
Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine, it is required for 18S rRNA maturation. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis, structure-function relationship, overview. Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Nep1 changes rRNA structure upon binding, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position
physiological function
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Nep1 is essential for psi119 pseudouridine methylation, but is not required for acp-modification. Nep1 has a dual function, as psi1191-methyltransferase and ribosome assembly factor
physiological function
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the nucleolar essential protein Nep1 is an important trans-acting factor in the 90S preribosome. Nep1 methylates the hypermodified psi1191 base of 18S rRNA and has an additional essential function during ribosome biogenesis, i.e. in 40S subunit synthesis. Utp30 and Nep1 act together during pre-ribosomal complex formation and, along with Rps18, provide the surface for the Rps19 assembly to the 90S pre-ribosome
physiological function
-
the rRNA modifications are thought to play a role in modulation of the three-dimensional structure of RNA and in fine-tuning its interactions with other RNAs or proteins
physiological function
the rRNA modifications are thought to play a role in modulation of the three-dimensional structure of RNA and in fine-tuning its interactions with other RNAs or proteins
physiological function
the rRNA modifications are thought to play a role in modulation of the three-dimensional structure of RNA and in fine-tuning its interactions with other RNAs or proteins
physiological function
-
the rRNA modifications are thought to play a role in modulation of the three-dimensional structure of RNA and in fine-tuning its interactions with other RNAs or proteins. The protein has an essential function in ribosomal biogenesis which directly or indirectly interferes with a methylation reaction during the early steps of pre-rRNA processing necessary for the generation of 40S ribosomal subunits
physiological function
-
the rRNA modifications are thought to play a role in modulation of the three-dimensional structure of RNA and in fine-tuning its interactions with other RNAs or proteins
-
physiological function
-
the rRNA modifications are thought to play a role in modulation of the three-dimensional structure of RNA and in fine-tuning its interactions with other RNAs or proteins. The protein has an essential function in ribosomal biogenesis which directly or indirectly interferes with a methylation reaction during the early steps of pre-rRNA processing necessary for the generation of 40S ribosomal subunits
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physiological function
-
Nep1 is a genuine rRNA methyltransferase and is essential for ribosome biogenesis
-
physiological function
-
the rRNA modifications are thought to play a role in modulation of the three-dimensional structure of RNA and in fine-tuning its interactions with other RNAs or proteins
-
additional information
active site structure and ligand binding, overview
additional information
Candida albicans NEP1 heterologously complements the essential phenotype in a Saccharomyces cerevisiae nep1 deletion mutant
additional information
-
Candida albicans NEP1 heterologously complements the essential phenotype in a Saccharomyces cerevisiae nep1 deletion mutant
additional information
-
genome-wide yeast screen to uncover synthetic interactions of DELTAnep1
additional information
-
human NEP1 heterologously complements the essential phenotype in a Saccharomyces cerevisiae nep1 deletion mutant
additional information
-
active site structure and ligand binding, overview
-
additional information
-
Candida albicans NEP1 heterologously complements the essential phenotype in a Saccharomyces cerevisiae nep1 deletion mutant
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). NEP1_YEAST
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
252
0
27895
Swiss-Prot
other Location (Reliability: 2)
A0A0Y9VN91_PLABE
231
0
26791
TrEMBL
other Location (Reliability: 1)
A0A077Y4L1_PLAYE
232
0
26965
TrEMBL
other Location (Reliability: 1)
A0A2P6RTL3_ROSCH
55
0
6430
TrEMBL
other Location (Reliability: 3)
A0A422PN61_9TRYP
272
0
28764
TrEMBL
Mitochondrion (Reliability: 5)
A0A1Z5JI76_FISSO
274
0
30291
TrEMBL
other Location (Reliability: 1)
A0A1C3KP59_9APIC
230
0
26621
TrEMBL
other Location (Reliability: 1)
A0A8B6CCF9_MYTGA
225
0
25067
TrEMBL
other Location (Reliability: 3)
A0A084G8F1_PSEDA
125
0
13879
TrEMBL
Mitochondrion (Reliability: 5)
A0A1A8VQU9_PLAMA
230
0
26722
TrEMBL
other Location (Reliability: 1)
A0A5B7AMM9_DAVIN
276
0
31221
TrEMBL
other Location (Reliability: 3)
A0A077TMT0_PLACH
231
0
26854
TrEMBL
other Location (Reliability: 1)
A0A6J8BLN5_MYTCO
225
0
25097
TrEMBL
other Location (Reliability: 3)
A0A1G4GTG1_PLAVI
231
0
26683
TrEMBL
other Location (Reliability: 1)
A0A0J9E9X7_9RHOB
315
0
33792
TrEMBL
-
A0A2P6QY71_ROSCH
114
0
12166
TrEMBL
other Location (Reliability: 2)
A0A0F8AZD1_CERFI
261
0
29067
TrEMBL
other Location (Reliability: 5)
A0A422QBX5_9TRYP
263
0
29765
TrEMBL
other Location (Reliability: 4)
A0A1L3KZ32_9HYPH
338
0
36610
TrEMBL
-
A0A8J5B480_9ASCO
258
0
28835
TrEMBL
other Location (Reliability: 1)
A0A1C3KLP1_PLAMA
230
0
26740
TrEMBL
other Location (Reliability: 1)
A0A5K1UNI0_PLAKH
231
0
26755
TrEMBL
other Location (Reliability: 1)
A0A8J8WJJ2_9EURO
257
0
28570
TrEMBL
other Location (Reliability: 3)
A0A384KSP2_PLAKH
231
0
26755
TrEMBL
other Location (Reliability: 1)
A0A422NAI1_TRYRA
292
0
31199
TrEMBL
other Location (Reliability: 5)
A0A3R7KPC0_TRYRA
273
0
30704
TrEMBL
other Location (Reliability: 3)
A0A1J1H1R2_PLARL
227
0
26452
TrEMBL
other Location (Reliability: 1)
A0A653GWU5_9APIC
229
0
26657
TrEMBL
other Location (Reliability: 1)
A0A1C6XKY8_PLACH
231
0
26854
TrEMBL
other Location (Reliability: 1)
A0A8K1DDA2_9ARCH
222
0
25130
TrEMBL
-
A0A2S5BBH5_9BASI
316
0
33424
TrEMBL
other Location (Reliability: 2)
A0A1D3KXY0_9APIC
229
0
26477
TrEMBL
other Location (Reliability: 1)
W1QJ52_OGAPD
Ogataea parapolymorpha (strain ATCC 26012 / BCRC 20466 / JCM 22074 / NRRL Y-7560 / DL-1)
256
0
28175
TrEMBL
other Location (Reliability: 2)
A0A8J5B6X1_9ASCO
258
0
28835
TrEMBL
other Location (Reliability: 1)
Q8IB49_PLAF7
279
0
32757
TrEMBL
other Location (Reliability: 1)
A0A812DF85_SEPPH
170
1
18990
TrEMBL
Secretory Pathway (Reliability: 1)
A0A1Z5KB49_FISSO
275
0
30505
TrEMBL
other Location (Reliability: 1)
A0A3G2S0G5_9BASI
364
0
39977
TrEMBL
other Location (Reliability: 1)
A0A7R8CPT4_LEPSM
154
0
16957
TrEMBL
other Location (Reliability: 3)
NEP1_ARCFU
Archaeoglobus fulgidus (strain ATCC 49558 / DSM 4304 / JCM 9628 / NBRC 100126 / VC-16)
219
0
25308
Swiss-Prot
-
NEP1_METJA
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
205
0
24082
Swiss-Prot
other Location (Reliability: 3)
NEP1_CANGA
Candida glabrata (strain ATCC 2001 / CBS 138 / JCM 3761 / NBRC 0622 / NRRL Y-65)
229
0
25596
Swiss-Prot
Secretory Pathway (Reliability: 5)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
additional information
dimer
-
three-dimensional structure of the Nep1 dimer, overview
additional information
Nep1 monomer and dimer structures, detailed overview
additional information
-
Nep1 monomer and dimer structures, detailed overview
-
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant detagged wild-type and selenomethionine-labeled afNep1 dimer bound to S-adenosyl homocysteine, X-ray diffraction structure determination and analysis at 1.45-2.0 A resolution
Nep1 in its free form and bound to S-adenosylhomocysteine or the antibiotic and general methyltransferase inhibitor sinefungin, hanging drop vapour diffusion method, 10-15 mg/ml protein in 100 mM BisTris-propane buffer, pH 9.5, 6-10% PEG 400, 30-48% glycerol, and 0-400 mM trimethylamine-N-oxide, at 4°C, crystals of the selenomethionine containing protein in the presence or absence of a 3fold excess of S-adenosyl-L-homocysteine are grown under similar conditions at 16°C, X-ray diffraction structure determination and analysis at 2.2 A, 2.15 A, and 2.25 A resolution, respectively
recombinant detagged scNep1 dimer bound to S-adenosyl homocysteine and in complexes with RNA, i.e. one molecule and two molecules of cognate RNA, X-ray diffraction structure determination and analysis at 1.80-1.90 A and at 3.00 A resolution, respectively
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D90G
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a mutation in yeast Nep1 equivalent to the Bowen-Conradi syndrome, BCS, mutation in the human Nep1. Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro
additional information
additional information
construction of a heterozygous CaDnep1/CaNEP1 strain CAE8 after replacement of one CaNEP1 wild-type allele with a CaURA3 marker and introduction of GFP-open reading frame driven by the methionine/cysteine-repressible CaMET3 promoter in front of the gene. Without high concentrations of methionine and cysteine in the medium, the resulting strain is viable, but addition of 2.5 mM methionine and 2.5 mM cysteine strongly impairs growth
additional information
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construction of a heterozygous CaDnep1/CaNEP1 strain CAE8 after replacement of one CaNEP1 wild-type allele with a CaURA3 marker and introduction of GFP-open reading frame driven by the methionine/cysteine-repressible CaMET3 promoter in front of the gene. Without high concentrations of methionine and cysteine in the medium, the resulting strain is viable, but addition of 2.5 mM methionine and 2.5 mM cysteine strongly impairs growth
additional information
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construction of a heterozygous CaDnep1/CaNEP1 strain CAE8 after replacement of one CaNEP1 wild-type allele with a CaURA3 marker and introduction of GFP-open reading frame driven by the methionine/cysteine-repressible CaMET3 promoter in front of the gene. Without high concentrations of methionine and cysteine in the medium, the resulting strain is viable, but addition of 2.5 mM methionine and 2.5 mM cysteine strongly impairs growth
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additional information
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complementation of a ScDnep1 strain with the human Nep1 (C2F) protein, the HsNEP1 open reading frame is expressed with the yeast inducible/repressible GAL1 promoter and the resulting plasmid (pGALHsNEP1) is transformed into the heterozygous ScDnep1/ScNEP1 strain CEN.SR679. After sporulation and tetrad analysis, ScDnep1 segregants complemented by the pGAL-HsNEP1 are only viable with galactose
additional information
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complementation of a ScDnep1 strain with the human Nep1 (C2F) protein, the HsNEP1 open reading frame is expressed with the yeast inducible/repressible GAL1 promoter and the resulting plasmid (pGALHsNEP1) is transformed into the heterozygous ScDnep1/ScNEP1 strain CEN.SR679. After sporulation and tetrad analysis, ScDnep1 segregants complemented by the pGAL-HsNEP1 are only viable with galactose
additional information
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lethal phenotype of a DELTAnep1 deletion, overexpression of RPS19B and also deletions within genes DELTAnop6 and DELTAtma23, respectively, which both encode small, positively charged fungal-specific proteins, suppress the DELTAnep1 growth deficiency. Identification of DELTanep1-specific genetic interactions, polysome profiles and phenotypes, overview. DELTAAutp30 enhances the phenotype of DELTAnep1 and is a multi-copy suppressor
additional information
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complementation of a ScDnep1 strain with the human Nep1 (C2F) protein, the HsNEP1 open reading frame is expressed with the yeast inducible/repressible GAL1 promoter and the resulting plasmid (pGALHsNEP1) is transformed into the heterozygous ScDnep1/ScNEP1 strain CEN.SR679. After sporulation and tetrad analysis, ScDnep1 segregants complemented by the pGAL-HsNEP1 are only viable with galactose
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additional information
construction of a heterozygous CaDnep1/CaNEP1 strain CAE8 after replacement of one CaNEP1 wild-type allele with a CaURA3 marker and introduction of GFP-open reading frame driven by the methionine/cysteine-repressible CaMET3 promoter in front of the gene. Without high concentrationsof methionine and cysteine in the medium, the resulting strain is viable, but addition of 2.5 mM methionine and 2.5 mM cysteine strongly impairs growth
additional information
-
construction of a heterozygous CaDnep1/CaNEP1 strain CAE8 after replacement of one CaNEP1 wild-type allele with a CaURA3 marker and introduction of GFP-open reading frame driven by the methionine/cysteine-repressible CaMET3 promoter in front of the gene. Without high concentrationsof methionine and cysteine in the medium, the resulting strain is viable, but addition of 2.5 mM methionine and 2.5 mM cysteine strongly impairs growth
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
native enzyme partially from subcellular fractions by sucrose density gradient fractionation
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recombinant His-tagged Nep1 from Escherichia coli strain M15(pREP4) by nickel affinity chromatography
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recombinant His6-tagged Nep1 from Escherichia coli strain RosettaTM-DE3-pLysS by nickel affinity chromatography, cleavage of the tag by TEV protease, and two steps of gel filtration to over 99% purity
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recombinant N-terminally His6-tagged wild-type and D90G mutant Nep1 from Escherichia coli strain XL1-blue by nickel affinity chromatography and gel filtration
recombinant Nep1 by cation exchange chromatography and gel filtration
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recombinant Nep1p from Escherichia coli strain BL21(DE3) by ion exchange chromatography and gel filtration
recombinant wild-type and selenomethionine-labeled His6-tagged Nep1 from Escherichia coli strain RosettaTM-DE3-pLysS by nickel affinity chromatography, cleavage of the tag by TEV protease, and two steps of gel filtration to over 99% purity
recombinant N-terminally His6-tagged wild-type and D90G mutant Nep1 from Escherichia coli strain XL1-blue by nickel affinity chromatography and gel filtration
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recombinant N-terminally His6-tagged wild-type and D90G mutant Nep1 from Escherichia coli strain XL1-blue by nickel affinity chromatography and gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of His6-tagged Nep1 in Escherichia coli strain RosettaTM-DE3-pLysS
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expression of wild-type and selenomethionine-labeled His6-tagged Nep1 in Escherichia coli strain RosettaTM-DE3-pLysS
gene NEP1, DNA and amino acid sequence determination and analysis
gene NEP1, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3)
gene NEP1, DNA and amino acid sequence determination and analysis, sequence comparisons, expression of the GFP-tagged ScNEP1 in enzyme-deficient Saccharomyces cerevisiae strain CEN.SR679 in the nucleus
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gene NEP1, expression of N-terminally His6-tagged wild-type and D90G mutant Nep1 in Escherichia coli strain XL1-blue
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gene NEP1, recombinant expression of His-tagged Nep1 in Escherichia coli strain M15(pREP4)
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gene NEP1, stable expression of GFP-tagged wild-type and D86G mutant Nep1 in 293T cells under control of the tetracycline-inducible promoter, expression of N-terminally His6-tagged wild-type and D90G mutant Nep1 in Escherichia coli strain XL1-blue
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
S-adenosylmethionine suppression of Nep1, suppression of mutant ScDELTAsnr35 nep1-1ts is independent of psi1191 methylation
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Eschrich, D.; Buchhaupt, M.; Koetter, P.; Entian, K.D.
Nep1p (Emg1p), a novel protein conserved in eukaryotes and archaea, is involved in ribosome biogenesis
Curr. Genet.
40
326-338
2002
Saccharomyces cerevisiae, Homo sapiens, [Candida] glabrata (Q96UP2), Candida albicans (Q9P8P7), Candida albicans, [Candida] glabrata CBS 138 (Q96UP2), Saccharomyces cerevisiae CEN.PK2, Candida albicans RM1000 (Q9P8P7)
brenda
Taylor, A.B.; Meyer, B.; Leal, B.Z.; Koetter, P.; Schirf, V.; Demeler, B.; Hart, P.J.; Entian, K.D.; Woehnert, J.
The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site
Nucleic Acids Res.
36
1542-1554
2008
Methanocaldococcus jannaschii (Q57977), Methanocaldococcus jannaschii DSM 2661 (Q57977)
brenda
Wurm, J.; Meyer, B.; Bahr, U.; Held, M.; Frolow, O.; Koetter, P.; Engels, J.; Heckel, A.; Karas, M.; Entian, K.; Woehnert, J.
The ribosome assembly factor Nep1 responsible for Bowen-Conradi syndrome is a pseudouridine-N1-specific methyltransferase
Nucleic Acids Res.
38
2387-2398
2010
Homo sapiens, Methanocaldococcus jannaschii
brenda
Meyer, B.; Wurm, J.P.; Koetter, P.; Leisegang, M.S.; Schilling, V.; Buchhaupt, M.; Held, M.; Bahr, U.; Karas, M.; Heckel, A.; Bohnsack, M.T.; Woehnert, J.; Entian, K.D.
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 psi1191 in yeast 18S rRNA
Nucleic Acids Res.
39
1526-1537
2011
Homo sapiens, Saccharomyces cerevisiae
brenda
Thomas, S.R.; Keller, C.A.; Szyk, A.; Cannon, J.R.; Laronde-Leblanc, N.A.
Structural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis
Nucleic Acids Res.
39
2445-2457
2011
Saccharomyces cerevisiae, Archaeoglobus fulgidus (O29524)
brenda
Schilling, V.; Peifer, C.; Buchhaupt, M.; Lamberth, S.; Lioutikov, A.; Rietschel, B.; Koetter, P.; Entian, K.D.
Genetic interactions of yeast NEP1 (EMG1), encoding an essential factor in ribosome biogenesis
Yeast
29
167-183
2012
Saccharomyces cerevisiae
brenda
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