Isolation and characterization of a nucleolar 2'-O-methyltransferase from Ehrlich ascites tumor cells

A 2'-O-methyltransferase that transfers the methyl group from S-adenosylmethionine to the 2'-hydroxyl group of ribose moieties of RNA has been purified from Ehrlich ascites tumor cell nucleoli. The partially purified enzyme is devoid of other RNA methylase activities and is free of ribonucleases. The enzyme has optimal activity in tris(hydroxymethyl)aminomethane buffer, pH 8.0, in the presence of 0.4 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, and 50 mM KCl, and has an apparent Km for S-adenosylmethionine of 0.44 microM. Gel filtration studies of this enzyme gave a Stokes radius of 43 A. Sedimentation velocity measurements in glycerol gradients yield an S20,w of 8.0 S. From these values, a native molecular weight of 145,000 was calculated. The enzyme catalyzes the methylation of synthetic homoribopolymers as well as 18S and 28S rRNA; however, poly(C) is the preferred synthetic substrate, and preference for unmethylated sequences of rRNA was observed. For each RNA substrate examined, only methylation of the 2'-hydroxyl group of the ribose moieties was detected.     

The specificity of interaction between S-adenosyl-L-methionine and a nucleolar 2'-O-methyltransferase

The structural features of S-adenosyl-L-methionine (SAM)3 required for optimal binding to a nucleolar 2'-O-methyltransferase were elucidated using various analogs of SAM with modifications of the amino acid, sugar, sulfonium center, and base portions of the molecule. Equilibrium binding constants for SAM and each analog were determined by a nitrocellulose filter binding assay. To ensure the chiral and chemical purity of the 3H-labeled SAM used in the binding experiments, a cation-exchange HPLC procedure was developed to separate degradation products of SAM such as adenine and 5'-deoxy-5'-methylthioadenosine, as well as to separate the (S,S)-SAM from the biologically inactive (R,S)-SAM stereoisomer. Results from these studies demonstrated that S-adenosyl-L-homocysteine, a product of the methyltransferase reaction, bound equally as well as (S,S)-SAM, indicating that neither the charge nor the methyl group at the sulfonium center of (S,S)-SAM is essential for maximal binding. Other modifications of the sulfonium center demonstrated that a sulfur to carbon atom replacement had little effect on binding affinity, whereas substituting an ethyl group for the methyl group greatly reduced the binding affinity. In addition, the chirality at the sulfonium center was important. The naturally occurring S-chiral form had a 10-fold higher binding affinity than the R-chiral form. No significant stereospecificity was observed relative to the chiral alpha-carbon of the methionine moiety in SAM. The alpha-amino group of methionine and the 6-amino group of adenine were both required for maximal binding, while the loss of the 2'-hydroxyl group on the ribose moiety was not. Taken together, these results defined some of the specific geometric and functional group requirements which affect the specificity of interaction between S-adenosyl-L-methionine and the nucleolar 2'-O-methyltransferase.     

The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity.

We have evidence that the open reading frame previously denoted spoU is necessary for tRNA (Gm18) 2'-O-methyltransferase activity. The spoU gene is located in the gmk-rpoZ-spoT-spoU-recG operon at 82 minutes on the Escherichia coli chromosome. The deduced amino acid sequence of spoU shows strong similarities to previously characterized 2'-O-methyltransferases. Comparison of the nucleoside modification pattern of hydrolyzed tRNA, 16S rRNA and 23S rRNA from wild-type and spoU null mutants showed that the modified nucleoside 2'-O-methylguanosine (Gm), present in a subset of E. coli tRNAs at residue 18, is completely absent in the spoU mutant, suggesting that spoU encodes tRNA (Gm18) 2'-O-methyltransferase. Nucleoside modification of 16S and 23S rRNA was unaffected in the spoU mutant. Insertions in the downstream recG gene did not affect RNA modification. Absence of Gm18 in tRNA does not influence growth rate under the tested conditions and does not interfere with activity of the SupF amber suppressor, a suppressor tRNA that normally has the Gm18 modification. We suggest that the spoU gene be renamed trmH (tRNA methylation).     

Aurora-B regulates RNA methyltransferase NSUN2

Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G(1) phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis.     

Biogenesis of small nucleolar ribonucleoproteins

Eukaryotic cells contain a very complex population of small nucleolar RNAs. They function, as small nucleolar ribonucleoproteins, in pre-ribosomal RNA processing reactions, and also guide methylation and pseudouridylation of ribosomal RNA, spliceosomal small nuclear RNAs, and possibly other cellular RNAs. Synthesis of small nucleolar RNAs frequently follows unusual strategies. Some newly discovered brain-specific small nucleolar RNAs of unknown function are encoded in introns of tandemly repeated units, expression of which is paternally imprinted. Recent studies of the protein components and factors participating in small nucleolar ribonucleoprotein assembly have revealed interesting connections with other classes of cellular ribonucleoproteins such as spliceosomal small nuclear ribonucleoproteins and telomerase. Cajal bodies emerge as nuclear structures important for the biogenesis and function of small nucleolar ribonucleoproteins.     

Metal determines efficiency and substrate specificity of the nuclear NUDIX decapping proteins X29 and H29K (Nudt16)

The Xenopus X29 protein was identified by its high affinity binding to U8 small nucleolar RNA, a small nucleolar RNA required for ribosome biogenesis. X29 and its human homologue H29K (Nudt16) are nuclear nucleoside diphosphatase proteins localized within foci in the nucleolus and nucleoplasm. These proteins can remove m(7)G and m(227)G caps from RNAs, rendering them substrates for 5'-3' exonucleases for degradation in vivo. Here, a more complete characterization of these metal-dependent decapping proteins demonstrates that the metal identity determines both the efficiency of decapping and the RNA substrate specificity. In Mg(+2) the proteins hydrolyze the 5' cap from only one RNA substrate: U8 small nucleolar RNA. However, in the presence of Mn(+2) or Co(+2) all RNAs are substrates and the decapping efficiency is higher. The x-ray crystal structure of X29 facilitated structure-based mutagenesis. Mutation of single amino acids coordinating metal in the active site yielded mutant proteins confirming essential residues. In vitro assays with purified components are consistent with a lack of protein turnover, apparently due to an inability of the protein to release the decapped RNA, implicating critical in vivo interacting factors. Collectively, these studies indicate that the metal that binds the X29/H29K proteins in vivo may determine whether these decapping proteins function solely as a negative regulator of ribosome biogenesis or can decap a wider variety of nuclear-limited RNAs. With the potential broader RNA substrate specificity, X29/H29K may be the nuclear counterparts of the cytoplasmic decapping machinery, localized in specialized bodies involved in RNA decay.     

Metal resistance in yeast mediated by the expression of a maize 20S proteasome alpha subunit

A novel 72 nt small nucleolar RNA (snoRNA) called U87 was found in rat liver cells. This RNA possesses the features of C/D box snoRNA family: boxes C, D', C', D, and 11 nt antisense element complementary to 28S ribosomal RNA (rRNA). The vast majority of C/D box snoRNAs direct site-specific 2'-O-ribose methylation of rRNAs. U87 RNA is suggested to be involved in 2'-O-methylation of a G(3468) residue in 28S rRNA. U87 RNA was detected in different mammalian species with slight length variability. Rat and mouse U87 RNA gene was characterized. Unlike the majority of C/D box snoRNAs U87 RNA lacks the terminal stem required for snoRNA processing. However, U87 gene is flanked by 7 bp inverted repeats potentially able to form a terminal stem in U87 RNA precursor.     

AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana

In all eukaryotes, C/D small nucleolar ribonucleoproteins (C/D snoRNPs) are essential for methylation and processing of ribosomal RNAs. They consist of a box C/D small nucleolar RNA (C/D snoRNA) associated with four highly conserved nucleolar proteins. Recent data in HeLa cells and yeast have revealed that assembly of these snoRNPs is directed by NUFIP protein and other auxiliary factors. Nevertheless, the precise function and biological importance of NUFIP and the other assembly factors remains unknown. In plants, few studies have focused on RNA methylation and snoRNP biogenesis. Here, we identify and characterise the AtNUFIP gene that directs assembly of C/D snoRNP. To elucidate the function of AtNUFIP in planta, we characterized atnufip mutants. These mutants are viable but have severe developmental phenotypes. Northern blot analysis of snoRNA accumulation in atnufip mutants revealed a specific degradation of C/D snoRNAs and this situation is correlated with a reduction in rRNA methylation. Remarkably, the impact of AtNUFIP depends on the structure of snoRNA genes: it is essential for the accumulation of those C/D snoRNAs encoded by polycistronic genes, but not by monocistronic or tsnoRNA genes. We propose that AtNUFIP controls the kinetics of C/D snoRNP assembly on nascent precursors to overcome snoRNA degradation of aberrant RNPs. Finally, we show that AtNUFIP has broader RNP targets, controlling the accumulation of scaRNAs that direct methylation of spliceosomal snRNA in Cajal bodies.     

A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance

Eukaryotes regulate the effective dosage of their ribosomal RNA (rRNA) genes, expressing fewer than half of the genes at any one time. Likewise, genetic hybrids displaying nucleolar dominance transcribe rRNA genes inherited from one parent but silence the other parental set. We show that rRNA gene dosage control and nucleolar dominance utilize a common mechanism. Central to the mechanism is an epigenetic switch in which concerted changes in promoter cytosine methylation density and specific histone modifications dictate the on and off states of the rRNA genes. A key component of the off switch is HDT1, a plant-specific histone deacetylase that localizes to the nucleolus and is required for H3 lysine 9 deacetylation and subsequent H3 lysine 9 methylation. Collectively, the data support a model in which cytosine methylation and histone deacetylation are each upstream of one another in a self-reinforcing repression cycle.     

Three new small nucleolar RNAs that are psoralen cross-linked in vivo to unique regions of pre-rRNA.

We have recently described three novel human small nucleolar RNA species with unique nucleotide sequences, which were named E1, E2, and E3. The present article describes specific psoralen photocross-linking in whole HeLa cells of E1, E2, and E3 RNAs to nucleolar pre-rRNA. These small RNAs were cross-linked to different sections of pre-rRNA. E1 RNA was cross-linked to two segments of nucleolar pre-rRNA; one was within residues 697 to 1163 of the 5' external transcribed spacer, and the other one was between nucleotides 664 and 1021 of the 18S rRNA sequence. E2 RNA was cross-linked to a region within residues 3282 to 3667 of the 28S rRNA sequence. E3 RNA was cross-linked to a sequence between positions 1021 and 1639 of the 18S rRNA sequence. Primer extension analysis located psoralen adducts in E1, E2, and E3 RNAs that were enriched in high-molecular-weight fractions of nucleolar RNA. Some of these psoralen adducts might be cross-links of E1, E2, and E3 RNAs to large nucleolar RNA. Antisense oligodeoxynucleotide-targeted RNase H digestion of nucleolar extracts revealed accessible segments in these three small RNAs. The accessible regions were within nucleotide positions 106 to 130 of E1 RNA, positions 24 to 48 and 42 to 66 of E2 RNA, and positions 7 to 16 and about 116 to 122 of E3 RNA. Some of the molecules of these small nucleolar RNAs sedimented as if associated with larger structures when both nondenatured RNA and a nucleolar extract were analyzed.     

The U14 snoRNA is required for 2'-O-methylation of the pre-18S rRNA in Xenopus oocytes.

We have studied the role of the U14 small nucleolar RNA (snoRNA) in pre-rRNA methylation and processing in Xenopus oocytes. Depletion of U14 in Xenopus oocytes was achieved by co-injecting two nonoverlapping antisense oligonucleotides. Focusing on the earliest precursor, depletion experiments revealed that the U14 snoRNA is essential for 2'-O-ribose methylation at nt 427 of the 18S rRNA. Injection of U14-depleted oocytes with specific U14 mutant snoRNAs indicated that conserved domain B, but not domain A, of U14 is required for the methylation reaction. When the effect of U14 on pre-rRNA processing is assayed, we find only modest effects on 18S rRNA levels, and no effect on the type or accumulation of 18S precursors, suggesting a role for U14 in a step in ribosome biogenesis other than cleavage of the pre-rRNA. Xenopus U14 is, therefore, a Box C/D fibrillarin-associated snoRNA that is required for site-specific 2'-O-ribose methylation of pre-rRNA.     

Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing

2'-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C'/D' motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA-protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C'/D' motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA-rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.