Profiling of RNA ribose methylation in Arabidopsis thaliana

Eukaryotic rRNAs and snRNAs are decorated with abundant 2′-O-methylated nucleotides (Nm) that are predominantly synthesized by box C/D snoRNA-guided enzymes. In the model plant Arabidopsis thaliana, C/D snoRNAs have been well categorized, but there is a lack of systematic mapping of Nm. Here, we applied RiboMeth-seq to profile Nm in cytoplasmic, chloroplast and mitochondrial rRNAs and snRNAs. We identified 111 Nm in cytoplasmic rRNAs and 19 Nm in snRNAs and assigned guide for majority of the detected sites using an updated snoRNA list. At least four sites are directed by guides with multiple specificities as shown in yeast. We found that C/D snoRNAs frequently form extra pairs with nearby sequences of methylation sites, potentially facilitating the substrate binding. Chloroplast and mitochondrial rRNAs contain five almost identical methylation sites, including two novel sites mediating ribosomal subunit joining. Deletion of FIB1 or FIB2 gene reduced the accumulation of C/D snoRNA and rRNA methylation with FIB1 playing a bigger role in methylation. Our data reveal the comprehensive 2′-O-methylation maps for Arabidopsis rRNAs and snRNAs and would facilitate study of their function and biosynthesis.     

Synthetic polynucleotides as model substrates for ribosomal RNA processing

A nuclear exoribonuclease from Novikoff ascites cells was used to study the hydrolysis of single-stranded heteropolymers containing [¹⁴C]adenylic acid and either uridylic acid or cytidylic acid and heteropolymers of [¹⁴C]adenylic acid and one of the corresponding 2′-$\text{O}$-methylated nucleotides. The results of these studies indicate that both the rate and extent of hydrolysis are greatly inhibited by the presence of 2′-$\text{O}$-methylated nucleotides. Restriction of exonuclease activity by 2′-$\text{O}$-methylated nucleotides provides a possible mechanism for rRNA processing.     

Metabolic stability and inhibitory effect of O-methylated theaflavins on H2O2-induced oxidative damage in human HepG2 cells

Seven new O-methylated theaflavins (TFs) were synthesized by using O-methyltransferase from an edible mushroom. Using TFs and O-methylated TFs, metabolic stability in pooled human liver S9 fractions and inhibitory effect on H₂O₂-induced oxidative damage in human HepG2 cells were investigated. In O-methylation of theaflavin 3′-O-gallate (TF3′G), metabolic stability was potentiated by an increase in the number of introduced methyl groups. O-methylation of TF3,3′G did not affect metabolic stability, which was likely because of a remaining 3-O-galloyl group. The inhibitory effect on oxidative damage was assessed by measuring the viability of H₂O₂-damaged HepG2 cells treated with TFs and O-methylated TFs. TF3,3′G and O-methylated TFs increased cell viabilities significantly compared with DMSO, which was the compound vehicle (p?<?0.05), and improved to approximately 100%. Only TF3′G did not significantly increase cell viability. It was suggested that the inhibitory effect on H₂O₂-induced oxidative damage was potentiated by O-methylation or O-galloylation of TFs.     

Sequence analysis of small amounts of nonradioactive oligoribonucleotides containing ribose-methylated nucleosides by a combination of 3H- and 32P-labeling techniques

The oligonucleotides A-G-A-Cm-U and Gm-A-A-Y-A-ψ were used as model compounds to demonstrate how the complete nucleotide sequence of small amounts of nonradioactive oligoribonucleotides (0.2–0.3 nmol) can be derived by a combination of ³H-labeling procedures previously published and a new method for the characterization of 2′-O-methylated nucleosides based on enzymatic ³²P labeling. The newly developed method for the identification of ribose-methylated nucleosides entails ³²P labeling by [γ-³²P]ATP/polynucleotide kinase of the 5′-terminus of a ribonuclease T₂-stable 2′-O-methylated dinucleotide derived from the polyribonucleotide, conversion of the labeled dinucleotide to the ³²P-labeled 2′-O-methylated nucleoside 5′-monophosphate, and identification of the monophosphate by its chromatographic properties on a polyethyleneimine-cellulose thin layer. The novel method is simple, fast, and sensitive and, at present, represents the only way by which ribose-methylated nucleosides can be analyzed in small amounts (0.01 nmol) of nonradioactive oligonculeotides or RNA.     

Characterisation of the U83 and U84 small nucleolar RNAs: two novel 2'-O-ribose methylation guide RNAs that lack complementarities to ribosomal RNAs

In eukaryotic cells, the site-specific 2′-O-ribose methy-lation of ribosomal RNAs (rRNAs) and the U6 spliceosomal small nuclear RNA (snRNA) is directed by small nucleolar RNAs (snoRNAs). The C and D box-containing 2′-O-methylation guide snoRNAs select the correct substrate nucleotide through formation of a long 10–21 bp interaction with the target rRNA and U6 snRNA sequences. Here, we report on the characterisation of two novel mammalian C/D box snoRNAs, called U83 and U84, that contain all the elements that are essential for accumulation and function of 2′-O-methylation guide snoRNAs. However, in contrast to all of the known 2′-O-methylation guide RNAs, the human, mouse and pig U83 and U84 snoRNAs feature no antisense elements complementary to rRNA or U6 snRNA sequences. The human U83 and U84 snoRNAs are not associated with maturing nucleolar pre-ribosomal particles, suggesting that they do not function in rRNA biogenesis. Since artificial substrate RNAs complementary to the evolutionarily conserved putative substrate recognition motifs of the U83 and U84 snoRNAs were correctly 2′-O-methy-lated in the nucleolus of mouse cells, we suggest that the new snoRNAs act as 2′-O-methylation guides for cellular RNAs other then rRNAs and the U6 snRNA.     

A simple method of the preparation of 2′-O-Methyladenosine Methylation of adenosine with methyl iodide in anhydrous alkaline medium

A simple and effective method of the methylation on the 2′-O position of adenosine is described. Adenosine is treated with CH₃I in an anhydrous alkaline medium at 0°C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2′-O or 3′-O position (total of 64%) and the side products are dimethylated adenosine (2′,3′-O-dimethyladenosi, 21%, and N⁶-2′-O-dimethyladenosine, 11%). The ratio of 2′-O- and 3′-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2′-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2′-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2′-O and 3′-O-methylated isomers. The overall yield of 2′-O-methyladenosine is 42%.     

The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans

During ribosomal RNA (rRNA) maturation, cleavages at defined sites separate the mature rRNAs from spacer regions, but the identities of several enzymes required for 18S rRNA release remain unknown. PilT N-terminus (PIN) domain proteins are frequently endonucleases and the PIN domain protein Utp24 is essential for early cleavages at three pre-rRNA sites in yeast (A0, A1 and A2) and humans (A0, 1 and 2a). In yeast, A1 is cleaved prior to A2 and both cleavages require base-pairing by the U3 snoRNA to the central pseudoknot elements of the 18S rRNA. We found that yeast Utp24 UV-crosslinked in vivo to U3 and the pseudoknot, placing Utp24 close to cleavage at site A1. Yeast and human Utp24 proteins exhibited in vitro endonuclease activity on an RNA substrate containing yeast site A2. Moreover, an intact PIN domain in human UTP24 was required for accurate cleavages at sites 1 and 2a in vivo, whereas mutation of another potential site 2a endonuclease, RCL1, did not affect 18S production. We propose that Utp24 cleaves sites A1/1 and A2/2a in yeast and human cells.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

Synthesis and characterization of modified nucleotides in the 970 hairpin loop of Escherichia coli 16S ribosomal RNA

The synthesis of the 6-O-DPC-2-N-methylguanosine (m²G) nucleoside and the corresponding 5′-O-DMT-2′-O-TOM-protected 6-O-DPC-2-N-methylguanosine phosphoramidite is reported [DPC, diphenyl carbamoyl; DMT, 4,4′-dimethoxytrityl; TOM, [(triisopropylsilyl)oxy]methyl]. The availability of the phosphoramidite allows for syntheses of hairpin RNAs with site-selective incorporation of 2-N-methylguanosine modification. Four 18-nt hairpin RNA analogues representing the 970-loop region (helix 31 or h31; U960–A975) of Escherichia coli 16S rRNA were synthesized with and without modifications in the loop region. Subsequently, stabilities and conformations of the singly and doubly modified RNAs were examined and compared with the corresponding unmodified RNA. Thermodynamic parameters and circular dichroism spectra are presented for the four helix 31 RNA analogues. Surprisingly, methylations in the loop region of helix 31 slightly destabilize the hairpin, which may have subtle effects on ribosome function. The hairpin construct is suitable for future ligand-binding experiments.     

Secondary structure maps of ribosomal RNA. II. Processing of mouse L-cell ribosomal RNA and variations in the processing pathway

Secondary structure mapping in the electron microscope was applied to ribosomal RNA and precusor ribosomal RNA molecules isolated from nucleoli and the cytoplasm of mouse L-cells. Highly reproducible loop patterns were observed in these molecules. The polarity of L-cell rRNA was determined by partial digestion with 3′-exonuclease. The 28 S region is located at the 5′-end of the 45 S rRNA precursor. Together with earlier experiments on labeling kinetics, these observations established a processing pathway for L-cell rRNA. The 45 S rRNA precursor is cleaved at the 3′-end of the 18 S RNA sequence to produce a 41 S molecule and a spacer-containing fragment (24 S RNA). The 41 S rRNA is cleaved forming mature 18 S rRNA and a 36 S molecule. The 36 S molecule is processed through a 32 S intermediate to the mature 28 S rRNA. This pathway is similar to that found in HeLa cells, except that in L-cells a 36 S molecule occurs in the major pathway and no 20 S precusor to 18 S RNA is found. The processing pathway and its intermediates in L-cells are analogous to those in Xenopus laevis, except for a considerable size difference in all rRNAs except 18 S rRNA.The arrangement of gene and transcribed spacer regions and of secondary structure loops, as well as the shape of the major loops were compared in L-cells, HeLa cell and Xenopus rRNA. The over-all arrangement of regions and loop patterns is very similar in the RNA from these three organisms. The shapes of loops in mature 28 S RNA are also highly conserved in evolution, but the shapes of loops in the transcribed spacer regions vary greatly. These observations suggest that the sequence complementarity that gives rise to this highly conserved secondary structure pattern may have some functional importance.     

Comparison of the nucleotide sequence of soybean 18S rRNA with the sequences of other small-subunit rRNAs

Summary We present the sequence of the nuclearencoded ribosomal small-subunit RNA from soybean. The soybean 18S rRNA sequence of 1807 nucleotides (nt) is contained in a gene family of approximately 800 closely related members per haploid genome. This sequence is compared with the ribosomal small-subunit RNAs of maize (1805 nt), yeast (1789 nt),Xenopus (1825 nt), rat (1869 nt), andEscherichia coli (1541 nt). Significant sequence homology is observed among the eukaryotic small-subunit rRNAs examined, and some sequence homology is observed between eukaryotic and prokaryotic small-subunit rRNAs. Conserved regions are found to be interspersed among highly diverged sequences. The significance of these comparisons is evaluated using computer simulation of a random sequence model. A tentative model of the secondary structure of soybean 18S rRNA is presented and discussed in the context of the functions of the various conserved regions within the sequence. On the basis of this model, the short basepaired sequences defining the four structural and functional domains of all 18S rRNAs are seen to be well conserved. The potential roles of other conserved soybean 18S rRNA sequences in protein synthesis are discussed.     

Changes in the Content of Modified Nucleotides in Wheat rRNA during Greening

The modified nucleotide content of the ribosomal RNAs in wheat is greatly influenced by light. The rRNAs of etiolated seedlings contain far fewer modified derivatives. The modified nucleotide composition characteristic of green plants develops gradually as a result of irradiation. In the course of the experiments changes in the state of modification of 5.8S and 18S rRNAs were examined during the greening of etiolated wheat seedlings. Three types of minor nucleotides, O^2′-methyladenosine, O^2′-methylguanosine and pseudouridine were found in the 5.8S rRNA of green wheat leaves, none of which was detected in etiolated wheat. The minor nucleotides appeared in the 5.8S rRNA only after 48 h irradiation. The sequences of 5.8S rDNA, TTS1, ITS2 and 18S rDNA were also determined and the presence of the hyper-modified nucleotide 1-methyl-3-(α-amino-α-carboxypropyl)-pseudouridine was detected in green wheat 18S rRNA. This minor component was not demonstrable in etiolated wheat 18S rRNA, but appeared after irradiation for 48 h. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flavonoid variation in some North Americna Saxifraga species

Eight species of Saxifraga representing sections Micranthes, Hirculus, Dactyloides and Xanthizoon were studied for their flavonoids (S. california, S. integrifolia, S. michauxii, S. ferruginea, S. eschscholtzii, S. hirculus, S. caespitosa and S. aizoides). The major compounds present in most species were kaempferol and quercetin monogluocosides and galactosides. Glucosides of kaempferol and quercetin predominate in the first four species listed, while galactosides of quercetin and myricetin are dominant in the lastthree. 3-O-Methyl- and 3,3′-di-O-methylquercetin were identified from S. californica and S. integrifolia. Saxifraga caespitosa synthesizes a complex mixture of O-methylated flavonols as well as a novel O-methylated dihydrokaempferol.Species pairs S. michauxii/S. ferruginea and S. californica/S.integrifolia exhibit close flavonoid similarity which may reflect their morphological similarities.     

Yeast Rrp8p,a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA

Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2′-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m¹A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m¹A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.     

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA (M_r = 2.7 × 10⁶) is much smaller than HeLa or L-cell 45 8 rRNA (M_r = 4.7 × 10⁶), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA.Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments.Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.