Evolution of the large-subunit ribosomal RNA binding site for protein L23/25

The region of the large-subunit rRNA encompassing the D7 divergent domain is organized within eukaryotes in a patchwork of short conservative secondary-structure features interspersed with more rapidly evolving sequences. It contains the attachment site of protein L25 (E. coli L23), which binds rRNA in the first stages of ribosome assembly, suggesting a crucial importance of this region in ribosome elaboration and functioning. A better understanding of its roles requires a good knowledge of its mode of structural variation during the course of evolution. With this aim, we sequenced the D7 region for 24 new invertebrate species belonging to annelids, molluscs, arthropods, and eight other deep-branching invertebrate phyla. Their comparison allowed us to propose refinements in previous eukaryotic folding models. A detailed analysis of the pattern of variation at each position both within the D7 region and along the L23/25 sequence by reference to previous heterologous binding experiments gives new insight into the rRNA-protein contacts. We identified in the D7 region and L23/25, respectively, six and five positions presenting a pattern of variation compatible with experimental results, three of which show coincident variations which support their possible involvement in the rRNA-L23/25 binding.      

Evolution of large subunit rRNA structure. The 3' terminal domain contains elements of secondary structure specific to major phylogenetic groups

Refined secondary structure models supported by phylogenetic evidence have been derived for the 3' terminal domain of large subunit rRNA (the region that exists as a separate 4.5 S molecular entity in chloroplast ribosomes) through a comparative analysis of all the pro- and eukaryotic sequences at present available. While several universally conserved features of secondary structure are found, a few diversified structural elements are also detected which are specific to one of the primary kingdoms, eubacteria, archaebacteria, or eukaryotes. Remarkably, some appear to be selectively preserved during the evolution of the primary kindgom, suggesting they represent functionally important structures. Thus, although the role of this 3' terminal domain in ribosomal function still remains unknown, its mode of sequence variation clearly points to a significant diversification of its function among the primary kindgoms.     

The complete nucleotide sequence of mouse 28S rRNA gene. Implications for the process of size increase of the large subunit rRNA in higher eukaryotes.

We have determined the complete nucleotide sequence (4712 nucleotides) of the mouse 28S rRNA gene. Comparison with all other homologs indicates that the potential for major variations in size during the evolution has been restricted to a unique set of a few sites within a largely conserved secondary structure core. The D (divergent) domains, responsible for the large increase in size of the molecule from procaryotes to higher eukaryotes, represent half the mouse 28S rRNA length. They show a clear potential to form self-contained secondary structures. Their high GC content in vertebrates is correlated with the folding of very long stable stems. Their comparison with the two other vertebrates, xenopus and rat, reveals an history of repeated insertions and deletions. During the evolution of vertebrates, insertion or deletion of new sequence tracts preferentially takes place in the subareas of D domains where the more recently fixed insertions/deletions were located in the ancestor sequence. These D domains appear closely related to the transcribed spacers of rRNA precursor but a sizable fraction displays a much slower rate of sequence variation.     

Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides, a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the “GPP” complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures.     

The structure of the yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18S ribosomal RNA gene from Saccharomyces cerevisiae.

The cloned 18 S ribosomal RNA gene from Saccharomyces cerevisiae have been sequenced, using the Maxam-Gilbert procedure. From this data the complete sequence of 1789 nucleotides of the 18 S RNA was deduced. Extensive homology with many eucaryotic as well as E. coli ribosomal small subunit rRNA (S-rRNA) has been observed in the 3'-end region of the rRNA molecule. Comparison of the yeast 18 S rRNA sequences with partial sequence data, available for rRNAs of the other eucaryotes provides strong evidence that a substantial portion of the 18 S RNA sequence has been conserved in evolution.     

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.     

Divergent evolution of a structural proteome: phenomenological models

We develop models of the divergent evolution of genomes; the elementary object of sequence dynamics is the protein structural domain. To identify patterns of organization that reflect mechanisms of evolution, we consider the individual genomes of many procaryote species, studying the arrangement of protein structural domains in the space of all polypeptide structures. We view the network of structural similarities as a graph, called the organismal Protein Domain Universe Graph (oPDUG); vertices represent types of structural domains and edges represent strong structural similarity. As observed before, each oPDUG is a highly nonrandom graph, as evidenced in the vertex degree distribution, which resembles a Pareto law (which has a power-law asymptotic). To explain this and other peculiar properties of the oPDUGs, we construct an evolving-graph model for the long-timescale evolutionary dynamics of oPDUGs, containing only divergent mechanisms of domain discovery. The model generates degree distributions (resembling Pareto laws) and clustering-coefficient distributions that are characteristic of the oPDUGs. In the infinite-graph limit, we analytically compute the exponent for specific biological parameters, as well as the complete phase diagram of the model, finding two distinct regimes of domain innovation dynamics. Thus, divergent evolutionary dynamics quantitatively explains the nonrandom organization of oPDUGs.     

Sequence and secondary structure of the central domain ofDrosophila 26S rRNA: A universal model for the central domain of the large rRNA containing the region in which the central break may happen

Summary An 890-bp sequence from the central region ofDrosophila melanogaster 26S ribosomal DNA (rDNA) has been determined and used in an extensive comparative analysis of the central domain of the large subunit ribosomal RNA (lrRNA) from prokaryotes, organelles, and eukaryotes. An alignment of these different sequences has allowed us to precisely map the regions of the central domain that have highly diverged during evolution. Using this sequence comparison, we have derived a secondary structure model of the central domain ofDrosophila 26S ribosomal RNA (rRNA). We show that a large part of this model can be applied to the central domain of lrRNA from prokaryotes, eukaryotes, and organelles, therefore defining a universal common structural core. Likewise, a comparative study of the secondary structure of the divergent regions has been performed in several organisms. The results show that, despite a nearly complete divergence in their length and sequence, a common structural core is also present in divergent regions. In some organisms, one or two of the divergent regions of the central domain are removed by processing events. The sequence and structure of these regions (fragmentation spacers) have been compared to those of the corresponding divergent regions that remain part of the mature rRNA in other species.     

An Empirical Analysis of mt 16S rRNA Covarion-Like Evolution in Insects: Site-Specific Rate Variation Is Clustered and Frequently Detected

The structural and functional analysis of rRNA molecules has attracted considerable scientific interest. Empirical studies have demonstrated that sequence variation is not directly translated into modifications of rRNA secondary structure. Obviously, the maintenance of secondary structure and sequence variation are in part governed by different selection regimes. The nature of those selection regimes still remains quite elusive. The analysis of individual bacterial models cannot adequately explore this topic. Therefore, we used primary sequence data and secondary structures of a mitochondrial 16S rRNA fragment of 558 insect species from 15 monophyletic groups to study patterns of sequence variation, and variation of secondary structure. Using simulation studies to establish significance levels of change, we found that despite conservation of secondary structure, the location of sequence variation within the conserved rRNA structure changes significantly between groups of insects. Despite our conservative estimation procedure we found significant site-specific rate changes at 56 sites out of 184. Additionally, site-specific rate variation is somewhat clustered in certain helices. Both results confirm what has been predicted from an application of non-stationary maximum likelihood models to rRNA sequences. Clearly, constraints on sequence variation evolve and leave footprints in the form of evolutionary plasticity in rRNA sequences. Here, we show that a better understanding of the evolution of rRNA sequences can be obtained by integrating both phylogenetic and structural information.     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

A re-evaluation of 12S ribosomal RNA variability in Drosophila pseudoobscura

Two recent studies have presented conflicting views on variation present within the 294 base third domain of the 12S rRNA gene in the genus Drosophila, and in D. pseudoobscura in particular. One study suggested that this gene is highly invariant across the genus, while another recovered 22 distinct haplotypes from 22 strains of D. pseudoobscura. We have sequenced this gene in numerous lines of D. pseudoobscura and its relatives, noting only two haplotypes in the third domain, and we failed to confirm any of the published sequences. Second, we note that the published sequence divergence between strains of D. pseudoobscura was as great as that documented between distantly related Drosophila species. Third, we show that the published polymorphisms of this region within D. pseudoobscura would disrupt the secondary structure of the resulting molecule. We conclude that the published 12S rRNA sequences of D. pseudoobscura do not accurately reflect variability of the functional gene, and that this gene is relatively invariant in D. pseudoobscura and D. persimilis.     

Localization of structural variations distinguishing I-Ak-related molecules to the alpha 1 and beta 1 domains

The structural variations that distinguish the A molecules encoded by wild-derived H-2 complexes which express Ak-related molecules have been localized into the alpha 1 and beta 1 domains by radiochemical sequence analyses of tryptic peptides. The A alpha subunits of B10.STC90 (Akv1) and W12A (Akv2) differ from those of B10.BR (Ak) in two adjacent tryptic peptides spanning positions 43 to 71 in the alpha 1 domain. The A beta subunit of W12A differs from that of B10.BR in two peptides spanning positions 26 to 29 and 95 to 106. Isoleucine and leucine residues present at positions 28 and 95, respectively, in the B10.BR A beta subunit are not found in the corresponding positions in W12A A beta subunits. Both of these A beta sequence variations are in the beta 1 domain. B10.STC90 A beta subunits are identical to those of W12A except for a structural variation in the beta 1 domain affecting the HPLC retention time of a peptide spanning positions 49 to 63. These results suggest that these A molecules are encoded by closely related class II gene alleles which have diversified by the accumulation of discrete mutations within the exons encoding the alpha 1 and beta 1 domains of the A molecule. Our previous functional analyses of these minor variant A molecules have demonstrated that they are readily distinguished with A molecule-specific alloreactive T lymphocytes. Together, these findings suggest that minor structural variations in the alpha 1 and beta 1 domains of the A molecule can dramatically modify the allodeterminants recognized by alloreactive T lymphocytes.     

Evolution of the secondary structures and compensatory mutations of the ribosomal RNAs of Drosophila melanogaster

This paper examines the effects of DNA sequence evolution on RNA secondary structures and compensatory mutations. Models of the secondary structures of Drosophila melanogaster 18S ribosomal RNA (rRNA) and of the complex between 2S, 5.8S, and 28S rRNAs have been drawn on the basis of comparative and energetic criteria. The overall AU richness of the D. melanogaster rRNAs allows the resolution of some ambiguities in the structures of both large rRNAs. Comparison of the sequence of expansion segment V2 in D. melanogaster 18S rRNA with the same region in three other Drosophila species and the tsetse fly (Glossina morsitans morsitans) allows us to distinguish between two models for the secondary structure of this region. The secondary structures of the expansion segments of D. melanogaster 28S rRNA conform to a general pattern for all eukaryotes, despite having highly divergent sequences between D. melanogaster and vertebrates. The 70 novel compensatory mutations identified in the 28S rRNA show a strong (70%) bias toward A-U base pairs, suggesting that a process of biased mutation and/or biased fixation of A and T point mutations or AT-rich slippage-generated motifs has occurred during the evolution of D. melanogaster rDNA. This process has not occurred throughout the D. melanogaster genome. The processes by which compensatory pairs of mutations are generated and spread are discussed, and a model is suggested by which a second mutation is more likely to occur in a unit with a first mutation as such a unit begins to spread through the family and concomitantly through the population. Alternatively, mechanisms of proofreading in stem-loop structures at the DNA level, or between RNA and DNA, might be involved. The apparent tolerance of noncompensatory mutations in some stems which are otherwise strongly supported by comparative criteria within D. melanogaster 28S rRNA must be borne in mind when compensatory mutations are used as a criterion in secondary-structure modeling. Noncompensatory mutation may extend to the production of unstable structures where a stem is stabilized by RNA- protein or additional RNA-RNA interactions in the mature ribosome. Of motifs suggested to be involved in rRNA processing, one (CGAAAG) is strongly overrepresented in the 28S rRNA sequence. The data are discussed both in the context of the forces involved with the evolution of multigene families and in the context of molecular coevolution in the rDNA family in particular.      

Analysis of the Secondary Structure of ITS1 in Pectinidae: Implications for Phylogenetic Reconstruction and Structural Evolution

It is at present difficult to accurately position gaps in sequence alignment and to determine substructural homology in structure alignment when reconstructing phylogenies based on highly divergent sequences. Therefore, we have developed a new strategy for inferring phylogenies based on highly divergent sequences. In this new strategy, the whole secondary structure presented as a string in bracket notation is used as phylogenetic characters to infer phylogenetic relationships. It is no longer necessary to decompose the secondary structure into homologous substructural components. In this study, reliable phylogenetic relationships of eight species in Pectinidae were inferred from the structure alignment, but not from sequence alignment, even with the aid of structural information. The results suggest that this new strategy should be useful for inferring phylogenetic relationships based on highly divergent sequences. Moreover, the structural evolution of ITS1 in Pectinidae was also investigated. The whole ITS1 structure could be divided into four structural domains. Compensatory changes were found in all four structural domains. Structural motifs in these domains were identified further. These motifs, especially those in D2 and D3, may have important functions in the maturation of rRNAs.     

Application of the secondary structure model of rRNA for phylogeny: D2-D3 expansion segments of the LSU gene of plant-parasitic nematodes from the family Hoplolaimidae Filipjev, 1934

Knowledge of rRNA structure is increasingly important to assist phylogenetic analysis through reconstructing optimal alignment, utilizing molecule features as an additional source of data and refining appropriate models of evolution of the molecule. We describe a procedure of optimization for alignment and a new coding method for nucleotide sequence data using secondary structure models of the D2 and D3 expansion fragments of the LSU-rRNA gene reconstructed for fifteen nematode species of the agriculturally important and diverse family Hoplolaimidae, order Tylenchida. Using secondary structure information we converted the original sequence data into twenty-eight symbol codes and submitted the transformed data to maximum parsimony analysis. We also applied the original sequence data set for Bayesian inference. This used the doublet model with sixteen states of nucleotide doublets for the stem region and the standard model of DNA substitution with four nucleotide states for loops and bulges. By this approach, we demonstrate that using structural information for phylogenetic analyses led to trees with lower resolved relationships between clades and likely eliminated some artefactual support for misinterpreted relationships, such as paraphyly of Helicotylenchus or Rotylenchus. This study as well as future phylogenetic analyses is herein supported by the development of an on-line database, NEMrRNA, for rRNA molecules in a structural format for nematodes. We also have developed a new computer program, RNAstat, for calculation of nucleotide statistics designed and proposed for phylogenetic studies.     

Phylogenetic calibration of the 5′ terminal domain of large rRNA achieved by determining twenty eucaryotic sequences

Summary Due to their high information content and their particular mode of variation, large rRNA molecules potentially represent powerful indicators of phylogenetic relationships. Even partial sequences may suffice to generate reliable estimations, provided they correspond to well-chosen portions of the molecule. We have systematically analyzed a specific portion of the large rRNA (the region extending over nearly 400 nucleotides from the 5 end) as a general index of eucaryotic phylogeny. By means of fast and direct rRNA sequencing, we have determined the sequence of this region for 20 additional eucaryotes, including several representatives of each vertebrate class, an invertebrate metazoan (mussel), a fungus (Schizosaccharomyces pombe), and three higher plants. Comparative treatment of these new data and previously reported rRNA sequences shows that this region can serve as an indicator of eucaryotic phylogeny for evaluating both long-range and short-range relationships. Its conservative domains appear to possess a rather uniform rate of nucleotide changes in all the eucaryotic lineages analyzed and the phylogenetic tree we derived agrees with classical views.     

Repeat arrays in cellular DNA related to the Epstein-Barr virus IR3 repeat.

We isolated clones and determined the sequence of portions of mouse and human cellular DNA which cross-hybridize strongly with the IR3 repetitive region of Epstein-Barr virus. The sequences were found to be tandem arrays of a simple sequence based on the triplet GGA, very similar to the IR3 repeat. The cellular repeats have distinct differences from the viral repeat region, however, and their sequences do not appear capable of being translated into a purely glycine-plus-alanine protein domain like the portion of the Epstein-Barr nuclear antigen coded by IR3. Although the relationship between IR3 and the cellular repeats is left unclear, the cellular repeats have many interesting features. The tandem arrays are about 1 to several kilobases long, much shorter than satellite tandem repeats and larger than other interspersed, tandem repeats. Each of the repeats is a distinct variation, perhaps diverged from a common sequence, (GGA)n. This family is present in the genomes of all species tested and appears to be a ubiquitous feature of all higher eucaryotic genomes.     

The Shwachman-Bodian-Diamond syndrome protein family is involved in RNA metabolism

A combination of structural, biochemical, and genetic studies in model organisms was used to infer a cellular role for the human protein (SBDS) responsible for Shwachman-Bodian-Diamond syndrome. The crystal structure of the SBDS homologue in Archaeoglobus fulgidus, AF0491, revealed a three domain protein. The N-terminal domain, which harbors the majority of disease-linked mutations, has a novel three-dimensional fold. The central domain has the common winged helix-turn-helix motif, and the C-terminal domain shares structural homology with known RNA-binding domains. Proteomic analysis of the SBDS sequence homologue in Saccharomyces cerevisiae, YLR022C, revealed an association with over 20 proteins involved in ribosome biosynthesis. NMR structural genomics revealed another yeast protein, YHR087W, to be a structural homologue of the AF0491 N-terminal domain. Sequence analysis confirmed them as distant sequence homologues, therefore related by divergent evolution. Synthetic genetic array analysis of YHR087W revealed genetic interactions with proteins involved in RNA and rRNA processing including Mdm20/Nat3, Nsr1, and Npl3. Our observations, taken together with previous reports, support the conclusion that SBDS and its homologues play a role in RNA metabolism.