5.8S-28S rRNA interaction and HMM-based ITS2 annotation

The internal transcribed spacer 2 (ITS2) of the nuclear ribosomal repeat unit is one of the most commonly applied phylogenetic markers. It is a fast evolving locus, which makes it appropriate for studies at low taxonomic levels, whereas its secondary structure is well conserved, and tree reconstructions are possible at higher taxonomic levels. However, annotation of start and end positions of the ITS2 differs markedly between studies. This is a severe shortcoming, as prediction of a correct secondary structure by standard ab initio folding programs requires accurate identification of the marker in question. Furthermore, the correct structure is essential for multiple sequence alignments based on individual structural features. The present study describes a new tool for the delimitation and identification of the ITS2. It is based on hidden Markov models (HMMs) and verifies annotations by comparison to a conserved structural motif in the 5.8S/28S rRNA regions. Our method was able to identify and delimit the ITS2 in more than 30 000 entries lacking start and end annotations in GenBank. Furthermore, 45 000 ITS2 sequences with a questionable annotation were re-annotated. Approximately 30 000 entries from the ITS2-DB, that uses a homology-based method for structure prediction, were re-annotated. We show that the method is able to correctly annotate an ITS2 as small as 58 nt from Giardia lamblia and an ITS2 as large as 1160 nt from humans. Thus, our method should be a valuable guide during the first and crucial step in any ITS2-based phylogenetic analysis: the delineation of the correct sequence. Sequences can be submitted to the following website for HMM-based ITS2 delineation: http://its2.bioapps.biozentrum.uni-wuerzburg.de.     

Sequence and secondary structure of mouse 28S rRNA 5''terminal domain. Organisation of the 5.8S-28S rRNA complex.

We present the sequence of the 5' terminal 585 nucleotides of mouse 28S rRNA as inferred from the DNA sequence of a cloned gene fragment. The comparison of mouse 28S rRNA sequence with its yeast homolog, the only known complete sequence of eukaryotic nucleus-encoded large rRNA (see ref. 1, 2) reveals the strong conservation of two large stretches which are interspersed with completely divergent sequences. These two blocks of homology span the two segments which have been recently proposed to participate directly in the 5.8S-large rRNA complex in yeast (see ref. 1) through base-pairing with both termini of 5.8S rRNA. The validity of the proposed structural model for 5.8S-28S rRNA complex in eukaryotes is strongly supported by comparative analysis of mouse and yeast sequences: despite a number of mutations in 28S and 5.8S rRNA sequences in interacting regions, the secondary structure that can be proposed for mouse complex is perfectly identical with yeast's, with all the 41 base-pairings between the two molecules maintained through 11 pairs of compensatory base changes. The other regions of the mouse 28S rRNA 5'terminal domain, which have extensively diverged in primary sequence, can nevertheless be folded in a secondary structure pattern highly reminiscent of their yeast' homolog. A minor revision is proposed for mouse 5.8S rRNA sequence.     

Nucleotide sequences of the 5.8S rRNAs of a mollusc and a porifer, and considerations regarding the secondary structure of 5.8S rRNA and its interaction with 28S rRNA

We report the primary structures of the 5.8 S ribosomal RNAs isolated from the sponge Hymeniacidon sanguinea and the snail Arion rufus. We had previously proposed (Ursi et al., Nucl. Acids Res. 10, 3517-3530 (1982)) a secondary structure model on the basis of a comparison of twelve 5.8 S RNA sequences then known, and a matching model for the interaction of 5.8 S RNA with 26 S RNA in yeast. Here we show that the secondary structure model can be extended to the 25 sequences presently available, and that the interaction model can be extended to the binding of 5.8 S RNA to the 5'-terminal domain of 28 S (26 S) RNA in three species.     

Primary and secondary structures of Tetrahymena and aphid 5.8S rRNAs: structural features of 5.8S rRNA which interacts with the 28S rRNA containing the hidden break

The Tetrahymena 5.8S rRNA is 154 nucleotides long, the shortest so far reported except for the split 5.8S rRNAs of Diptera (m5.8S plus 2S rRNA). In this molecule several nucleotides are deleted in the helix e (GC-rich stem) region. Upon constructing the secondary structure in accordance with "burp-gun" model, the Tetrahymena 5.8S rRNA forms a wide-open "muzzle" of the terminal regions due to both extra nucleotides and several unpaired bases. The aphid 5.8S rRNA consists of 161 nucleotides and can form stable helices in both terminal and helix e regions. As a whole, the secondary structure of Tetrahymena 5.8S rRNA resembles that of Bombyx 5.8S molecule while the aphid 5.8S rRNA shares several structural features with the HeLa 5.8S molecule. Likely, the 5.8S rRNA attached to the 28S rRNA with the hidden break differs in structure from those interacting with the 28S partners without the break. Nucleotide sequences of 5.8S rRNA in insects as well as in protozoans are not so conservative evolutionarily as in vertebrates.     

Effect of point mutations on 5.8S ribosomal ribonucleic acid secondary structure and the 5.8S--28S ribosomal ribonucleic acid junction

Naturally occurring differences in the nucleotide sequences of 5.8S ribosomal ribonucleic acids (rRNAs) from a variety of organisms have been used to study the role of specific nucleotides in the secondary structure and intermolecular interactions of this RNA. Significant differences in the electrophoretic mobilities of free 5.8S RNAs and the thermal stabilities of 5.8S--28S rRNA complexes were observed even in such closely related sequences as those of man, rat, turtle, and chicken. A single base transition from a guanylic acid residue in position 2 in mammalian 5.8S rRNA to an adenylic acid residue in turtle and chicken 5.8S rRNA results both in a more open molecular conformation and in a 5.8S--28S rRNA junction which is 3.5 degrees C more stable to thermal denaturation. Other changes such as the deletion of single nucleotides from either the 5' or the 3' terminals have no detectable effect on these features. The results support secondary structure models for free 5.8S rRNA in which the termini interact to various degrees and 5.8S--28S rRNA junctions in which both termini of the 5.8S molecule interact with the cognate high molecular weight RNA component.     

A 12 S precursor to 5.8 S rRNA associated with rat liver nucleolar 28 S rRNA

The pre-rRNA and rRNA components of rat and mouse liver nucleolar RNA were analysed. It was shown that upon denaturation, part of the 32 S pre-rRNA is converted into 28 S rRNA and 12 S RNA. The 12 S RNA from mouse (Mr, 0.36 X 10(6)) is larger than the one from rat (Mr, 0.32 X 10(6). The 12 S RNA chain is intact and resists denaturation treatment. The non-covalent binding of this RNA with nucleolar 28 S rRNA is stronger than that of 5.8 S rRNA with 28 S rRNA. Hybridization with a rat internal-transcribed spacer rDNA fragment identifies 12 S RNA as corresponding to the 5'-end non-conserved segment of 32 S pre-rRNA, including 5.8 S rRNA. The significance of the formation of a 12 S precursor to 5.8 S rRNA in the biogenesis of ribosomes in mammalian cells is discussed.     

Sequence and secondary structure of Drosophila melanogaster 5.8S and 2S rRNAs and of the processing site between them.

Drosophila melanogaster 5.8S and 2S rRNAs were end-labeled with 32p at either the 5' or 3' end and were sequenced. 5.8S rRNA is 123 nucleotides long and homologous to the 5' part of sequenced 5.8S molecules from other species. 2S rRNA is 30 nucleotides long and homologous to the 3' part of other 5.8S molecules. The 3' end of the 5.8S molecule is able to base-pair with the 5' end of the 2S rRNA to generate a helical region equivalent in position to the "GC-rich hairpin" found in all previously sequenced 5.8S molecules. Probing the structure of the labeled Drosophila 5.8S molecule with S1 nuclease in solution verifies its similarity to other 5.8S rRNAs. The 2S rRNA is shown to form a stable complex with both 5.8S and 26S rRNAs separately and together. 5.8S rRNA can also form either binary or ternary complexes with 2S and 26S rRNA. It is concluded that the 5.8S rRNA in Drosophila melanogaster is very similar both in sequence and structure to other 5.8 rRNAs but is split into two pieces, the 2S rRNA being the 3' part. 2S anchors the 5.8S and 26S rRNA. The order of the rRNA coding regions in the ribosomal DNA repeating unit is shown to be 18S - 5.8S - 2S - 26S. Direct sequencing of ribosomal DNA shows that the 5.8S and 2S regions are separated by a 28 nucleotide spacer which is A-T rich and is presumably removed by a specific processing event. A secondary structure model is proposed for the 26S-5.8S ternary complex and for the presumptive precursor molecule.     

Different mechanisms for pseudouridine formation in yeast 5S and 5.8S rRNAs

The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.     

Prediction of the Recognition Sites on 16S and 23S rRNAs from E.coli for the Formation of 16S-23S rRNA Complex

Abstract

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E.coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891–899 and 1195–1203 on 16S rRNA along with the corresponding complementary sites 1904–1912 and 760–768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630–638 on 16S rRNA with its complementary site 2031–2039 on 23S rRNA cannot be ruled out.     

Prediction of the recognition sites on 16S and 23S rRNAs from E. coli for the formation of 16S-23S rRNA complex

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E. coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891-899 and 1195-1203 on 16S rRNA along with the corresponding complementary sites 1904-1912 and 760-768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630-638 on 16S rRNA with its complementary site 2031-2039 on 23S rRNA cannot be ruled out.     

A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes

The organization of the 18S, 28S and 5.8S rRNA genes in the mouse has been elucidated by mapping with restriction endonucleases Eco RI, Hind III and Bam HI. Ribosomal DNA fragments were detected in electrophoretically fractionated digests of total nuclear DNA by in situ hybridization with radioiodinated rRNAs or with complementary RNA synthesized directly on rRNA templates. A map of the rDNA which includes 13 restriction sites was constructed from the sizes of rDNA fragments and their labeling by different probes The map indicates that the rRNA genes lie within remarkably large units of reiterated DNA, at least 44,000 base pairs long. At least two, and possibly four, classes of repeating unit can be distinguished, the heterogeneity probably residing in the very large nontranscribed spacer region. The 5.8S rRNA gene lies in the transcribed region between the 18S and 28S genes.     

Chemical modification studies and the secondary structure of HeLa cell 5.8S rRNA.

Various secondary structure models have been proposed for 5.8 S rRNA. In this paper HeLa cell 5.8 S rRNA is shown to possess several sites that are reactive to carbodiimide at 25 degrees, and other regions that are unreactive. Previous work has established the distribution of reactive and unreactive cytidine residues along the primary structure (11). The secondary structure model of Nazar et al. (7) is fully compatible with the chemical reactivity data whereas other models are partly incompatible. We conclude that the model of Nazar et al. provides the best approximation so far available to the conformation of isolated 5.8 S rRNA. Findings on the effect temperature on the chemical reactivity of different parts of the structure are summarized. The findings described in this paper should provide a basis for examining the specific interaction of 5.8 S rRNA with 28 s rRNA.     

Structural organization of ribosomal RNAs from Novikoff hepatoma. II. Characterization of possible binding sites of 5 S rRNA and 5.8 S rRNA to 28 S rRNA

Interrelationships among 5 S, 5.8 S, and 28 S rRNA were probed by methods employed in the accompanying report (Choi, Y. C. (1985) J. Biol. Chem. 260, 12769-12772). Two complexes were isolated from 20 S ribonucleoprotein (RNP) fraction and 60 S subunit. The 20 S RNP fraction was found to contain the 3'-340 nucleotide fragment (domain VII) in association with 5 S rRNA. The 60 S subunit contained a stable complex consisting of the 5'-upstream portion (4220-4462, domain VI and VII), the 3'-downstream portion (4463-4802, domain VII) of 3'-583 nucleotides fragment, and 5.8 S rRNA. By computer analysis and hybridization, the 5'-upstream portion was found to contain the 5.8 S rRNA contact site. By affinity chromatography, the 3'-downstream portion was found to contain the 5 S rRNA association site. Furthermore, by comparison with the secondary structure of 28 S rRNA proposed by Hadjiolov et al. (Hadjiolov, A. A., Georgiev, O. I., Nosikov, V. V., and Yavachev, L. P. (1984) Nucleic Acids Res. 12, 3677-3693), it was found that domain VII is capable of binding 5.8 S rRNA and 5 S rRNA juxtaposed to each other. Accordingly, a model was proposed to indicate that a possible contact site for 5.8 S rRNA is within the region surrounding the alpha-sarcin site (4333-4350) and is a possible association site of 5 S rRNA within the 3'-downstream portion (4463-4802) of the 3'-583 nucleotide fragment (4220-4802).     

Primary and secondary structure of 5.8S rRNA from the silkgland of Bombyx mori.

Nucleotide sequence of 5.8S rRNA of the silkworm, Bombyx mori has been determined by gel sequencing methods. The 5.8S rRNA was the longest so far reported, with the 5'-terminal sequence several nucleotides longer than those of the other organisms. Upon constructing the secondary structure in accordance with the "burp gun" model (12), the Bombyx 5.8S rRNA formed a wide-open "muzzle" due to several unpaired bases at the ends. The overall structure also appeared less stable with less G . C pairs and more unpaired bases than that of the HeLa 5.8S rRNA. These structural features may be essential for those 5.8S rRNAs which interact with 28S rRNAs containing the hidden break to form a stable complex.     

核糖体RNA二级结构对拓扑结构准确性的影响

探讨了核糖体小亚基二级结构对真菌系统发育分析的影响。对用不同方法构建的系统发育树进行比较,结果表明结合二级结构信息的分析方法较传统方法产生了更为合理的拓扑结构。二级结构信息除用于优化序列比对外,还需整合到核酸替代模型中;恰当的序列比对方法、进化模型和建树运算法则有助于更加准确地揭示类群之间的亲缘关系。     

Role of the 5.8S rRNA in ribosome translocation.

Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation.     

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.