The ribosome assembly factor Nep1 responsible for Bowen-Conradi syndrome is a pseudouridine-N1-specific methyltransferase

Abstract

Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen-Conradi syndrome-a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910-921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Psi in eukaryotic 18S rRNAs.

Figure 1.

3D structure of Nep1 and secondary structure and conservation of hypothetical Nep1 methyltransferase target sites in 18S rRNA. (A) Three-dimensional structure of the M. jannaschii Nep1 dimer in cartoon representation bound to the cofactor S-adenosylhomocysteine (3bbd). Insertion elements extending the SPOUT-class RNA methyltransferase core fold and unique to the Nep1 subfamily are colored gray in one monomer and red in the other monomer, respectively. Highly conserved arginine side chains implicated in RNA binding and Trp193 are shown in a stick representation. (B) Left: Secondary structure of a fragment of yeast 18S rRNA containing two examples of the yeast Nep1 binding RNA-consensus motif 5′-C/UUCAAC-3′ (red) in the proximity of putative methylation target sites. Modification sites are indicated as boxed nucleotides and the type of modification is given. Furthermore, the chemical structure of the hypermodified pseudouridine in position 1191 is shown in the inset. Right: Secondary structure and sequence of the same fragment of Methanocaldococcus jannaschii 16S rRNA with the location of the only conserved copy of the RNA consensus motif highlighted in red.

Figure 2.

Identification of sequence determinants required for high affinity binding by fluorescence quenching experiments and yeast three-hybrid library screening. (A) Fluorescence titration curve of MjNep1 with an RNA oligonucleotide containing a 5′-extension of the 5′-UUCAAC-3′ consensus motif (RNA 3, Table 1). (B) Fluorescence titration curves of MjNep1 with RNA oligonucleotides containing either 3′-end extensions (RNA 5, Table 1) or 5′-end and 3′-end extensions (RNA 7,8) and a pseudouridine modification at a position corresponding to nucleotide 914 in M. jannaschii 16S rRNA (RNA 8). (C) Results of the yeast three-hybrid screening against a focused library of 5′-NNNCAACNNN-3′ containing RNAs displayed as a sequence logo.

Figure 3.

RNA-methyltransferase activity of MjNep1. (A) MALDI mass spectra of a reaction mixture of S-adenosylmethionine, MjNep1 and high affinity RNAs containing a pseudouridine either at the position corresponding to nt 914 (highlighted in red) or scrambled to the preceding position. The formation of a reaction product with a 14 Da increase in mass corresponding to the addition of a methyl group is observable for 5′-GAUΨCAACGCC-3′ but not for 5′-GAΨUCAACGCC-3′.

Figure 4.

Identification of the modified nucleoside as N1-methylpseudouridine by RP-HPLC and NMR-spectroscopy. (A) HPLC-chromatograms of hydrolyzed and dephosphorylated RNA 12 prior to (top) and after treatment with MjNep1 in the presence of S-adenosylmethionine (middle). The signal of the pseudouridine nucleoside disappears and a novel signal (indicated by an asterisk) appears (middle) at a retention time very similar to that of commercially available N1-methylpseudouridine (bottom). (B) 2D-¹H,¹H-NOESY (top) and 1D-¹H- (middle) NMR-spectra of the modified nucleoside purified by preparative RP-HPLC from MjNep1 treated RNA 12 compared to a 1D ¹H-NMR-spectrum of commercially available N1-methylpseudouridine (bottom). NMR-signal assignments are indicated. Signals of an impurity are marked by asterisks. The section of the 2D-¹H,¹H-NOESY-spectrum (top) of the modified nucleoside reveals an NOE between the aromatic H6 proton of the pyrimdine ring and the methyl group. Such an NOE is only observable in N1-methylpseudouridine but not in N3-methylpseudouridine (see inset).

Figure 5.

Conserved pseudouridine dependent RNA-methyltransferase activity of human Nep1. MALDI mass spectra of oligonucleotides corresponding to nt 1245–1255 of human 18S rRNA incubated with HsNep1 and SAM containing either a Ψ (top and middle) or a U (bottom) residue at a position equivalent to nt 1248 modified to m1acp3-Ψ in human 18S rRNA. The appearance of a reaction product with an additional mass of 14 Da corresponding to an additional methyl group is only observed in the case of the pseudouridine containing oligonucleotide.

Figure 6.

Chemical shift mapping of the RNA-binding surface of MjNep1 for its high affinity substrates and orientation of the RNA in the binding pocket. (A) ¹H,¹⁵N-HSQC-spectra of uniformly ²H,¹⁵N-labeled MjNep1 in the absence (black) and the presence (red) of RNA 11 indicate the formation of a specific RNA–protein complex. (B) ¹H,¹⁵N-HSQC-spectra of selectively ¹⁵N-lysine labeled MjNep1 in the absence (black) and the presence (red) of RNA 11. (C) Distribution of lysines across the surface of the MjNep1 dimer (amino acids in blue and orange) and location of lysines affected by the addition of RNA 11 (highlighted in orange). Unaffected lysines are coloured blue and the bound cofactor SAH is coloured yellow. (D) Mapping of RNA 11 induced chemical shift changes in uniformly and selectively labeled MjNep1 samples on the surface of the MjNep1 dimer. Amino acids with significant chemical shift changes are coloured in orange, unaffected amino acids are colored in blue, amino acids with untraceable signals are coloured black and the bound cofactor SAH is colored yellow. (E) Localization of the 3′-end of the bound RNA by investigating differential chemical shift changes induced by RNA 11 and RNA 10 carrying an extra nucleotide at its 3′-end. Left: Overlay of ¹H,¹⁵N-HSQC-spectra of selectively ¹⁵N-arginine labeled free MjNep1 (black), MjNep1 (labelled with an *) bound to RNA 11 (red) and MjNep1 bound to the extended RNA 10 (blue). Two signals are only affected by the presence of RNA 10 indicating a location of the extra 3′-nucleotide close to R24 (red orange) and R27 (red orange) in the large irregularly structured extension loop (right). Arginine residues with signals affected by both RNAs are shown in orange. Those with chemical shifts unaffected by either RNA are coloured blue. (F) Localization of the 5′-end of the bound RNA using paramagnetic relaxation enhancement induced by spin labeled RNA. Left: Overlay of ¹H,¹⁵N-HSQC-spectra of selectively ¹⁵N-lysine labeled MjNep1 in the presence of an RNA containing a spin-label at position 5 of the 5′-terminal uridine residue (5′-UUCAACGCC-3′, the spin-labeled nucleotide is highlighted in italics) when the spin-label is oxidized and paramagnetic (black) and upon reduction to its diamagnetic state (red). Right: Mapping of the amino acids showing no signals in the presence of the oxidized spin-label due to spatial proximity to the 5′-end of the bound RNA on the structure of the MjNep1 dimer. Affected lysines are shown in orange, unaffected lysines are colored blue. The signals of K143 and K30 are too weak to be reliably tracked in these experiments.