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PubMed1986

Length variation in eukaryotic rRNAs: small subunit rRNAs from the protists Acanthamoeba castellanii and Euglena gracilis.

We have sequenced the region of the Acanthamoeba castellanii ribosomal RNA transcription unit which encodes the mature small subunit ribosomal RNA (SSU rRNA). It, like the SSU rRNA coding regions of Euglena gracilis and kinetoplastids, is approx. 30% larger than those reported from other eukaryotes. The extra nucleotides are present in highly variable regions of the rRNA genes. Direct sequence analysis of the corresponding variable regions in the rRNA of A. castellanii and E. gracilis demonstrates that the extra nucleotides are present in the mature rRNA; no post-transcriptional modification of the rRNAs occurs to reduce them to a size more typical of eukaryotes. The extra elements present in the rRNAs of these two organisms are not homologous; they have independent evolutionary origins.

Amoeba↗
PubMed1989

Ribonucleoside analysis by reversed-phase high-performance liquid chromatography.

Over the past fifteen years we have developed and refined the analytical chromatographic methodologies using reversed-phase high-performance liquid chromatography and UV-photodiode array detection (RPLC-UV) for the detection and measurement of the major and modified nucleosides in nucleic acids and biological fluids. RPLC-UV nucleoside analysis as it has now evolved is a powerful new research tool to aid investigators in the fields of biochemical and biomedical research. This RPLC-UV nucleoside method can resolve more than 65 nucleosides in a single analysis with "run-to-run" peak retention variations of less than 1%. A complete nucleoside composition can be obtained from as little as 0.5 micrograms RNA. Identification and confirmation of nucleosides can be made from the highly reproducible retention times and from the characteristic UV spectrum from a few picomoles (ca. 1 ng) of nucleoside. In this paper we introduce standard RPLC-UV methodologies for the analysis of nucleosides and nucleoside composition of RNAs. The chromatographic protocols and standard nucleoside columns are presented and the essential requirements necessary in the HPLC instrumentation are described. Three optimized RPLC systems were developed, each with particular emphasis placed on resolution, speed, or sensitivity. In addition, three unfractionated tRNAs were selected as sources of reference nucleosides and for assessment of the performance of the chromatography. From these tRNAs, a large array of nucleosides were characterized which are used in standardization and calibration of the method. Also discussed is the use of a diode-array detector for enhancement of the reliability of nucleoside identification and accuracy of measurement. An extended enzymatic hydrolysis protocol for the liberation of exotically modified nucleosides in tRNAs is also described. Chromatographic retention times and UV spectra for a large number of ribonucleosides are tabulated. The RPLC-UV ribonucleoside analytical protocols are capable of quantifying 31 nucleosides. Approximately 1 microgram of an isoaccepting tRNA, or 20 micrograms of unfractionated tRNA are needed for quantitative analysis. With this amount of tRNA, the percent relative error of measurement of the four major nucleosides is less than 2%, and for the modified nucleosides about 5%. As little as 0.2 micrograms of pure isoaccepting tRNA can be analyzed, but at the expense of precision as at this low sample size a 20-30% relative error for modified nucleosides is to be expected. For quantitation of the modified nucleosides in rRNA, which contains much less modification than tRNAs, 10-100 micrograms of sample are needed per injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Acquired resistance in Mycobacterium avium complex strains isolated from AIDS patients and beige mice during treatment with clarithromycin.

Clarithromycin has been reported to select clarithromycin resistant mutants of Mycobacterium avium complex (MAC) during treatment with clarithromycin in AIDS patients and beige mice. We selected resistant mutants in vitro at a frequency of 5 x 10(-9). Clarithromycin resistant strains of MAC isolated in AIDS patients and beige mice as well as derivatives selected in vitro had a unique pattern of acquired cross-resistance to macrolides and related antibiotics. In contrast, the pattern of resistance to non-macrolide antibiotics remained unchanged in clarithromycin resistant strains. A dramatic decrease in ribosome affinity for clarithromycin and erythromycin was found in clarithromycin resistant strains, but no mutation was found in the peptidyl domain of the 23S rRNA, indicating that another ribosomal modification is involved.

Acquired Immunodeficiency Syndrome↗
PubMed1979

A unique method utilizing antinucleotide antibodies for evaluating changes in the levels of modified nucleosides of tRNAs from crude extracts of whole cells.

The utilization of antibodies directed toward modified nucleosides in evaluating changes in the levels of certain modified nucleosides in transfer RNA is reported. Antibodies directed toward the N6-(delta 2-isopentenyl)adenosine modification were used in this model system with a mutant strain of Escherichia coli designated ipaA. The procedure is rapid, sensitive, and specific. In addition, it does not depend on the existence of an in vitro remodification system or any radiochemical labeling of the tRNA. By varying the extraction technique, the method could be applied to procaryotic or eukaryotic cell lines. The existence of antibodies specific for other nucleoside modifications makes this a system that is potentially applicable to a variety of deficiencies in the modification of both tRNA and rRNA.

Antibodies↗
PubMed2004

RMF inactivates ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel.

In gram-negative bacteria such as Escherichia coli, protein synthesis is suppressed by the formation of 100S ribosomes under stress conditions. The 100S ribosome, a dimer of 70S ribosomes, is formed by ribosome modulation factor (RMF) binding to the 70S ribosomes. During the stationary phase, most of the 70S ribosomes turn to 100S ribosomes, which have lost translational activity. This 100S formation is called the hibernation process in the ribosome cycle of the stationary phase. If stationary phase cells are transferred to fresh medium, the 100S ribosomes immediately go back to active 70S ribosomes, showing that inactive 100S <--> active 70S interconversion is a major system regulating translation activity in stationary phase cells. To elucidate the mechanisms of translational inactivation, the binding sites of RMF on 23S rRNA in 100S ribosome of E. coli were examined by a chemical probing method using dimethyl sulphate (DMS). As the results, the nine bases in 23S rRNA were protected from DMS modifications and the modification of one base was enhanced. Interestingly A2451 is included among the protected bases, which is thought to be directly involved in peptidyl transferase activity. We conclude that RMF inactivates ribosomes by covering the peptidyl transferase (PTase) centre and the entrance of peptide exit tunnel. It is surprising that the cell itself produces a protein that seems to inhibit protein synthesis in a similar manner to antibiotics and that it can reversibly bind to and release from the ribosome in response to environmental conditions.

Amino Acid Sequence↗
PubMed1987

RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes.

The modification reaction of 28 S rRNA in eukaryotic ribosomes by ricin A-chain was characterized. To examine whether ricin A-chain release any bases from 28 S rRNA, rat liver ribosomes were incubated with a catalytic amount of the toxin, and a fraction containing free bases and nucleosides was prepared from the postribosomal fraction of the reaction mixture by means of ion-exchange column chromatography. Thin-layer chromatographic analysis of this fraction revealed a release of 1 mol of adenine from 1 mol of ribosome. When the ribosomes or naked total RNAs were treated with ricin A-chain in the presence of [32P] phosphate, little incorporation of the radioactivity into 28 S rRNA was observed, indicating that the release is not mediated by phosphorolysis. Thus, considering together with the previous result (Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K. (1987) J. Biol. Chem. 262, 5908-5912), the results in the present experiments demonstrated that ricin A-chain inactivates the ribosomes by cleaving the N-glycosidic bond of A4324 of 28 S rRNA in a hydrolytic fashion.

Animals↗
PubMed2003

Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m6A methyltransferase ErmC'.

Methyltransferases (MTases) from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, still very little is known about the mechanism of rRNA:adenine-N(6) methylation. Only predictions regarding various aspects of this reaction have been made based on the analysis of the crystal structures of methyltransferase ErmC' (without the RNA) and their comparison with the crystallographic and biochemical data for better studied DNA:m(6)A MTases. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC', we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m(6)A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m(6)A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the "catalytic" motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC'. This somewhat surprising result is discussed in the light of the available structural data and in the phylogenetic context of the Erm family.

Adhesins, Bacterial↗
PubMed2004

Small nucleolar RNAs that guide modification in trypanosomatids: repertoire, targets, genome organisation, and unique functions.

Small nucleolar RNAs constitute a family of newly discovered non-coding small RNAs, most of which function in guiding RNA modifications. Two prevalent types of modifications are 2'-O-methylation and pseudouridylation. The modification is directed by the formation of a canonical small nucleolar RNA-target duplex. Initially, RNA-guided modification was shown to take place on rRNA, but recent studies suggest that small nuclear RNA, mRNA, tRNA, and the trypanosome spliced leader RNA also undergo guided modifications. Trypanosomes contain more modifications and potentially more small nucleolar RNAs than yeast, and the increased number of modifications may help to preserve ribosome function under adverse environmental conditions during the cycling between the insect and mammalian host. The genome organisation in clusters carrying the two types of small nucleolar RNAs, C/D and H/ACA-like RNAs, resembles that in plants. However, the trypanosomatid H/ACA RNAs are similar to those found in Archaea and are composed of a single hairpin that may represent the primordial H/ACA RNA. In this review we summarise this new field of trypanosome small nucleolar RNAs, emphasising the open questions regarding the number of small nucleolar RNAs, the repertoire, genome organisation, and the unique function of guided modifications in these protozoan parasites.

Animals↗
PubMed2000

Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry.

We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5' end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.

Base Sequence↗
PubMed2002

The structure of the RlmB 23S rRNA methyltransferase reveals a new methyltransferase fold with a unique knot.

In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2'O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2'O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.

Amino Acid Sequence↗
PubMed1976

Directed cleavage of ribopolynucleotides with nucleases restricted by multiple modification of substrate.

Simultaneous exhaustive modification of cytidine and uridine residues of rRNA with methoxyamine and sodium metabisulfite renders adjacent phosphodiester bonds resistant to pancreatic and T2 ribonucleases. Another method of T2 RNAase restriction is modification of cytidine with methoxyaminebisulfite followed by modification of guanosine residues with beta-ethoxy-alpha-ketobutyraldehyde. Mild alkaline treatment leads to demodification of uridine and guanosine residues leaving intact modified cytidine residues, thus providing a means of stepwise, directed cleavage of the polynucleotide. The series of combined cleavage procedures and methods of isolation of oligo(C), oligo(G) and oligopyrimidine tracts, as well as the procedure of selective cleavage at uridine residues elaborated in the course of the present studies may serve as a basis for more rational procedures of RNA sequencing.

Base Sequence↗
PubMed1997

Structural analyses of plastid-derived 16S rRNAs in holoparasitic angiosperms.

Higher-order structures have been constructed for plastid-encoded small-subunit (SSU, 16S), rRNAs from representatives of seven nonphotosynthetic holoparasitic angiosperm families: Apodanthaceae, Cynomoriaceae, Cytinaceae, Balanophoraceae, Hydnoraceae, Mitrastemonaceae, and Rafflesiaceae. Whereas most pairwise comparisons among angiosperms differ by 2-3% in substitutions, the 16S rRNAs of the holoparasites show an increasingly greater number of mutations: Cynomorium (7.3%), Cytinus (8.0%), Bdallophyton (12.7%), Mitrastema (14.9%), Hydnora (19.4%), Pilostyles (30.4%) and Corynaea (35.9%). Despite this high level of sequence variation, SSU structures constructed for all species except Pilostyles possess the typical complement of 50 helices (that contain numerous compensatory mutations) thereby providing indirect evidence supporting their functionality. Pilostyles, likely with the most unusual plastid 16S rRNA yet documented, lacks four major helices and contains lengthy insertions for four others. Sequences of products generated via RT-PCR show that these structural modifications are present on a mature (transcribed) rRNA. The trend toward increasing numbers of base substitutions in the holoparasites is accompanied by a marked increase in A+U content of the rRNA. This 'A/T drift' phenomenon of rDNA is especially apparent in Corynaea whose SSU rDNA sequence is 72% A+T. A comparison of Cytinus to tobacco showed that substitution rates appear to be dependent upon the composition of neighboring bases. Transversions represented 26% of the mutations when flanking bases were G or C whereas transversions increased to 36% when the flanking bases were A to T. The underlying molecular mechanism associated with these high substitution rates is presently unknown, however, relaxation of selection pressure on ribosome function resulting in altered DNA replication and/or repair systems may be involved.

Base Composition↗
PubMed1999

Structure and biogenesis of small nucleolar RNAs acting as guides for ribosomal RNA modification.

Maturation of pre-ribosomal RNA (pre-rRNA) in eukaryotic cells takes place in the nucleolus and involves a large number of cleavage events, which frequently follow alternative pathways. In addition, rRNAs are extensively modified, with the methylation of the 2'-hydroxyl group of sugar residues and conversion of uridines to pseudouridines being the most frequent modifications. Both cleavage and modification reactions of pre-rRNAs are assisted by a variety of small nucleolar RNAs (snoRNAs), which function in the form of ribonucleoprotein particles (snoRNPs). The majority of snoRNAs acts as guides directing site-specific 2'-O-ribose methylation or pseudouridine formation. Over one hundred RNAs of this type have been identified to date in vertebrates and the yeast Saccharomyces cerevisiae. This number is readily explained by the findings that one snoRNA acts as a guide usually for one or at most two modifications, and human rRNAs contain 91 pseudouridines and 106 2'-O-methyl residues. In this article we review information about the biogenesis, structure and function of guide snoRNAs.

Animals↗
PubMed2005

Mutations in MTO2 related to tRNA modification impair mitochondrial gene expression and protein synthesis in the presence of a paromomycin resistance mutation in mitochondrial 15 S rRNA.

Nuclear gene(s) have been shown to modulate the phenotypic expression of mitochondrial DNA mutations. We report here the identification and characterization of the yeast nuclear gene MTO2 encoding an evolutionarily conserved protein involved in mitochondrial tRNA modification. Interestingly, mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA at the very conservative site for human deafness-associated 12 S rRNA A1491G and C1409T mutations. Furthermore, the overall rate of mitochondrial translation was markedly reduced in a yeast mto2 strain in the wild type mitochondrial background, whereas mitochondrial protein synthesis was almost abolished in a yeast mto2 strain carrying the C1409G allele. The other interesting feature of mto2 mutants is the defective expression of mitochondrial genes, especially CYTB and COX1, but only when coexisting with the C1409G allele. These data strongly indicate that a product of MTO2 functionally interacts with the decoding region of 15 S rRNA, particularly at the site of the C1409G or A1491G mutation. In addition, we showed that yeast and human Mto2p localize in mitochondria. The isolated human MTO2 cDNA can partially restore the respiratory-deficient phenotype of yeast mto2 cells carrying the C1409G mutation. These functional conservations imply that human MTO2 may act as a modifier gene, modulating the phenotypic expression of the deafness-associated A1491G or C1409T mutation in mitochondrial 12 S rRNA.

Alleles↗
PubMed2004

RNA structure and function in C/D and H/ACA s(no)RNPs.

From archaea to humans, C/D- and H/ACA-type small ribonucleoprotein particles play key roles in crucial RNA processing events. Various such particles are required for pre-rRNA cleavage steps and/or for chemical modification of rRNAs, spliceosomal small nuclear RNAs, tRNAs and perhaps even mRNAs. Each C/D-type particle contains a small RNA possessing conserved C and D, as well as related C' and D', sequence motifs, whereas each H/ACA-type particle contains a small RNA featuring conserved H and ACA sequence elements. Recently published studies highlight the importance of sequence and structural elements of these RNAs in the localization, activity and assembly of the ribonucleoprotein particles. A novel sequence element, the Cajal body box, found at the apex of stem structures within a subset of H/ACA small RNAs, mediates the specific retention of particles containing these elements inside nucleoplasmic Cajal bodies. Two highly conserved elements, the m1 and m2 boxes, have been identified in the 3' stem of the atypical H/ACA snR30/U17 RNAs. These conserved sequence elements are necessary for early pre-rRNA cleavage events and consequently for mature 18S rRNA production. Finally, convincing evidence has been provided that the conserved C and D sequence motifs of C/D-type small RNAs fold into a helix-bulge-helix structure, called a kink-turn, that provides a platform for assembly of C/D-type ribonucleoprotein particles.

Animals↗
PubMed2006

Identification of nucleotides in E. coli 16S rRNA essential for ribosome subunit association.

The ribosome consists of two unequal subunits, which associate via numerous intersubunit contacts. Medium-resolution structural studies have led to grouping of the intersubunit contacts into 12 directly visualizable intersubunit bridges. Most of the intersubunit interactions involve RNA. We have used an RNA modification interference approach to determine Escherichia coli 16S rRNA positions that are essential for the association of functionally active 70S ribosomes. Modification of the N1 position of A702, A1418, and A1483 with DMS, and of the N3 position of U793, U1414, and U1495 with CMCT in 30S subunits strongly interferes with 70S ribosome formation. Five of these positions localize into previously recognized intersubunit bridges, namely, B2a (U1495), B2b (U793), B3 (A1483), B5 (A1418), and B7a (A702). The remaining position displaying interference, U1414, forms a base pair with G1486, which is a part of bridge B3. We contend that these five intersubunit bridges are essential for reassociation of the 70S ribosome, thus forming the functional core of the intersubunit contacts.

Centrifugation, Density Gradient↗
PubMed2002

Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria.

Posttranscriptional modifications were mapped in helices 90-92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90-92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-directed RNase H digestion. A novel method of mapping modifications by analysis of short, nucleotide-specific, RNase digestion fragments with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was utilized. The MALDI-MS data were complemented by two primer extension techniques using reverse transcriptase. One technique utilizes decreasing concentrations of deoxynucleotide triphosphates to map 2'-O-ribose methylations. In the other, the rRNA is chemically modified, followed by mild alkaline hydrolysis to map pseudouridines (psis). A total of 10 posttranscriptionally methylated nucleotides and 6 psis were detected in the five organisms. Eight of the methylated nucleotides and one psi have not been reported previously. The distribution of modified nucleotides and their locations on the surface of the ribosomal peptidyl transferase cleft suggests functional importance.

Bacillus subtilis↗