Analysis of the Primary Sequence and Secondary Structure of the Unusually Long SSU rRNA of the Soil Bug, Armadillidium vulgare

The complete nucleotide sequence of the SSU rRNA gene from the soil bug, Armadillidium vulgare (Crustacea, Isopoda), was determined. It is 3214 bp long, with a GC content of 56.3%. It is not only the longest SSU rRNA gene among Crustacea but also longer than any other SSU rRNA gene except that of the strepsipteran insect, Xenos vesparum (3316 bp). The unusually long sequence of this species is explained by the long sequences of variable regions V4 and V7, which make up more than half of the total length. RT-PCR analysis of these two regions showed that the long sequences also exist in the mature rRNA and sequence simplicity analysis revealed the presence of slippage motifs in these two regions. The putative secondary structure of the rRNA is typical for eukaryotes except for the length and shape variations of the V2, V4, V7, and V9 regions. Each of the V2, V4, and V7 regions was elongated, while the V9 region was shortened. In V2, two bulges, located between helix 8 and helix 9 and between helix 9 and helix 10, were elongated. In V4, stem E23-3 was dramatically expanded, with several small branched stems. In V7, stem 43 was branched and expanded. Comparisons with the unusually long SSU rRNAs of other organisms imply that the increase in total length of SSU rRNA is due mainly to expansion in the V4 and V7 regions. Received: 2 March 1999 / Accepted: 22 July 1999     

Evolution of Hypervariable Regions, V4 and V7, of Insect 18S rRNA and Their Phylogenetic Implications

We compared primary and secondary structures of V4 (helices E23-2 to E23-5) and V7 (helix 43) regions of 18S rRNAs in insects and the other three major arthropod groups (crustaceans, myriapods, and chelicerates) known so far. We found that the lengths of primary sequences and the shapes of secondary structures of these two hypervariable regions of insect 18S rRNA even at infraclass levels are phylogenetically informative and reflect major steps in insect evolution. The long sequence insertion and bifurcated shape of helices E23-2 to E23-5 in the V4 region are unique synapomorphic characters for winged insects (Pterygota). The long sequence insertion and expanded stem length of helix 43 in the V7 region are synapomorphic characters for holometabolous insects which conduct complete metamorphosis. The strongly conserved secondary structures suggest the possibility that these hypervariable regions may be related with certain important cellular functions unknown thus far. The comparison with insect fossil records revealed that the pterygote synapomorphy (V4) and the holometabolous synapomorphy (V7) were established prior to the acquisition of insect wings (flight system) and prior to the development of complete metamorphosis, respectively. These synapomorphies have been also relatively stable over at least 300 Myr and 280 Myr, respectively as well. It implies that the expansion events of the V4 and V7 regions have not occurred simultaneously but independently at different periods during the insect evolution. Then this suggests that V4 and V7 regions are not functionally correlated as recently suggested by Crease and Coulbourn.     

A comparison of the crystal structures of eukaryotic and bacterial SSU ribosomal RNAs reveals common structural features in the hypervariable regions

While the majority of the ribosomal RNA structure is conserved in the three major domains of life--archaea, bacteria, and eukaryotes, specific regions of the rRNA structure are unique to at least one of these three primary forms of life. In particular, the comparative secondary structure for the eukaryotic SSU rRNA contains several regions that are different from the analogous regions in the bacteria. Our detailed analysis of two recently determined eukaryotic 40S ribosomal crystal structures, Tetrahymena thermophila and Saccharomyces cerevisiae, and the comparison of these results with the bacterial Thermus thermophilus 30S ribosomal crystal structure: (1) revealed that the vast majority of the comparative structure model for the eukaryotic SSU rRNA is substantiated, including the secondary structure that is similar to both bacteria and archaea as well as specific for the eukaryotes, (2) resolved the secondary structure for regions of the eukaryotic SSU rRNA that were not determined with comparative methods, (3) identified eukaryotic helices that are equivalent to the bacterial helices in several of the hypervariable regions, (4) revealed that, while the coaxially stacked compound helix in the 540 region in the central domain maintains the constant length of 10 base pairs, its two constituent helices contain 5+5 bp rather than the 6+4 bp predicted with comparative analysis of archaeal and eukaryotic SSU rRNAs.     

DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita

Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles. However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision. The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5′- and 3′-ends were localized by nuclease S1 mapping, leading to a size of 3277 nt. The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C. The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases. The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs. Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3′-part of the SSU rRNA. This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility. This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision.     

The origin and evolution of variable-region helices in V4 and V7 of the small-subunit ribosomal RNA of branchiopod crustaceans

We sequenced the V4 and V7 regions of the small-subunit ribosomal RNA (SSU rRNA) from 38 species of branchiopod crustaceans (e.g., Artemia, Daphnia, Triops) representing all eight extant orders. Ancestral large-bodied taxa in the orders Anostraca, Notostraca, Laevicaudata, and Spinicaudata (limnadiids and cyzicids) possess the typical secondary structure in these regions, whereas the spinicaudatan Cyclestheria and all of the cladocerans (Anomopoda, Ctenopoda, Onychopoda, and Haplopoda) possess three unique helices. Although the lengths and primary sequences of the distal ends of these helices are extremely variable, their locations, secondary structures, and primary sequences at the proximal end are conserved, indicating that they are homologous. This evidence supports the classical view that Cladocera is a monophyletic group and that the cyclestheriids are transitional between spinicaudatans and cladocerans. The single origin and persistence since the Permian of the unique cladoceran helices suggests that births and deaths of variable region helices have been rare. The broad range of sequence divergences observed among the cladoceran helices permitted us to make inferences about their evolution. Although their proximal ends are very GC-biased, there is a significant negative correlation between length and GC content due to an increasing proportion of U at their distal ends. Slippage-like processes occurring at unpaired nucleotides or bulges, which are very U-biased, are associated with both helix origin and runaway length expansion. The overall GC contents and lengths of V4 and V7 are highly correlated. More surprisingly, the lengths of these SSU rRNA variable regions are also highly correlated with the length of the large-subunit rRNA expansion segment, D2, indicating that mechanisms affecting length variation do so both across single genes and across genes in the rRNA gene family.     

The secondary structure of the unusually long 18S ribosomal RNA of the myxozoan Sphaerospora truttae and structural evolutionary trends in the Myxozoa

The nearly complete 18S rRNA sequence of the myxozoan parasite Sphaerospora truttae shows an extraordinary length (2,552bp) in comparison with other myxozoans and with metazoans in general (average 1,800-1,900bp). The sequence shows nucleotide insertions in most variable regions of the 18S rRNA (V2, V4, V5 and V7), with especially large expansion segments in V4 and V7. In the myxozoans, nucleotide insertions and specific secondary structures in these regions of the gene were found to be strongly related to large scale phylogenetic clustering and thus with the invertebrate host type. Whereas expansion segments were generally found to be absent in the malacasporeans and the clade of primary marine myxozoan species, they occur in all taxa of the clade containing freshwater species, where they showed a consistent secondary structure throughout. The longest expansion segments occur in S. truttae, Sphaerospora elegans and Leptotheca ranae, which represent a clade that has emerged after the malacosporeans and before the radiation of all other myxozoan genera. These three species demonstrate structural links to the malacosporeans as well as other unique features. A smaller number of nucleotide insertions in different subhelices and specific secondary structures appear to have evolved independently in two marine genera, i.e. Ceratomyxa and Parvicapsula. The secondary structural elements of V4 and V7 of the myxozoan 18S rRNAs were found to be highly informative and revealed evolutionary trends of various regions of the gene hitherto unknown, since previous analyses have been based on primary sequence data excluding these regions. Furthermore, the unique features of the V4 region in S. truttae allowed for the design of a highly specific PCR assay for this species.     

A discontinuous small subunit ribosomal RNA in Tetrahymena pyriformis mitochondria

We show here that in the mitochondria of Tetrahymena pyriformis, the small subunit (SSU) rRNA is discontinuous, being comprised of two separate components which we term "alpha" (a novel low molecular weight RNA, approximately equal to 200 nucleotides long) and "beta" (a previously described 14 S RNA). The SSU alpha rRNA has been sequenced in its entirety; it represents the immediate 5'-terminal domain of conventional SSU rRNA. The sequences at the ends of the SSU beta rRNA have also been determined; they show that this molecule corresponds to the 3'-terminal 7/8 of conventional SSU rRNA. A 2.5-kilobase pair XbaI restriction fragment of T. pyriformis mitochondrial DNA which contains the SSU alpha and SSU beta rRNA genes was cloned and its complete nucleotide sequence was determined. This revealed that the genes encoding the two segments of SSU rRNA are separated by a 54-base pair (A + T)-rich spacer. The alpha and beta sequences can be fitted to a generalized secondary structure model for eubacterial 16 S rRNA, with the two RNA species associating through long range interactions to form base-paired regions characteristic of SSU rRNA. In this model, the spacer is situated in a region of pronounced primary and secondary structural variation among SSU rRNAs. The significance of these findings with respect to rRNA biosynthesis and processing and the possible evolutionary relationship between spacers and variable regions in rRNA genes is discussed.     

A comparison of the solution structures and conformational properties of the somatic and oocyte 5S rRNAs of Xenopus laevis

The secondary and tertiary structures of Xenopus oocyte and somatic 5S rRNAs were investigated using chemical and enzymatic probes. The accessibility of both RNAs towards single-strand specific nucleases (T1, T2, A and S1) and a helix-specific ribonuclease from cobra venom (RNase V1) was determined. The reactivity of nucleobase N7, N3 and N1 positions towards chemical probes was investigated under native (5 mM MgCl2, 100 mM KCl, 20 degrees C) and semi-denaturing (1 mM EDTA, 20 degrees C) conditions. Ethylnitrosourea was used to identify phosphates not reactive towards alkylation under native conditions. The results obtained confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. The chemical reactivity data indicate that loops C and D are involved in a number of tertiary interactions, and loop E folds into an unusual secondary structure. A comparison of the data obtained for the two types of Xenopus 5S rRNA indicates that the conformations of the oocyte and somatic 5S rRNAs are very similar. However, the data obtained with nucleases under native conditions, and chemical probes under semi-denaturing conditions, reveal that helices III and IV in the somatic 5S rRNA are less stable than the same structures in oocyte 5S rRNA. Using chimeric 5S rRNAs, it was possible to demonstrate that the relative resistance of oocyte 5S rRNA to partial denaturation in 4 M urea is conferred by the five oocyte-specific nucleotide substitutions in loop B/helix III. In contrast, the superior stability of oocyte 5S rRNA in the presence of EDTA is related to a single C substitution at position 79.     

A case study to estimate the applicability of secondary structures of SSU‐rRNA gene in taxonomy and phylogenetic analyses of ciliates

Phylogenetic studies of ciliates are mainly based on the primary structure information of the nuclear genes. Some regions of the small subunit ribosomal RNA (SSU‐rRNA) gene have distinctive secondary structures, which have demonstrated value as phylogenetic/taxonomic characters. In the current work, we predict the secondary structures of four variable regions (V2, V4, V7 and V9) in the SSU‐rRNA gene of 45 urostylids. Structure comparisons indicate that the V4 region is the most effective in revealing interspecific relationships, while the V9 region appears suitable at the family level or higher. The V2 region also offers some taxonomic information, but is too conserved to reflect phylogenetic relationships at the family or lower level, at least for urostylids. The V7 region is the least informative. We constructed several phylogenetic trees, based on the primary sequence alignment and based on an improved alignment according to the secondary structures. The results suggest that including secondary structure information in phylogenetic analyses provides additional insights into phylogenetic relationships. Using urostylid ciliates as an example, we show that secondary structure information results in a better understanding of their relationships, for example generic relationships within the family Pseudokeronopsidae.     

The unusually long small subunit ribosomal RNA gene found in amitochondriate amoeboflagellate Pelomyxa palustris: its rRNA predicted secondary structure and phylogenetic implication

In order to ascertain a phylogenetic position of the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be 3502 bp long. The predicted secondary structure of its rRNA includes at least 16 separate expansion zones located in all the variable regions (V1-V9), as well as in some conservative gene regions. Most insertions are represented by sequences of low complexity that have presumably arisen by a slippage mechanism. Relatively conservative, uniformly positioned motifs contained in regions V4 and V7, as well as in some others, made it possible to perform folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P. palustris tends to cluster with amitochondriate and secondary lost mitochondria amoebae and amoeboflagellates Entamoeba, Endolimax nana, and Phreatamoeba balamuthi, comprising together with them and aerobic lobose amoebae Vannella, Acanthamoeba, Balamuthia, and Hartmannella a monophyletic cluster. Another pelobiont, Mastigamoeba invertens, does not belong to this cluster. No specific similarity was discovered between the SSU rRNA of P. palustris and amitochondriate taxa of 'Archezoa': Diplomonada, Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA does not occupy a basal position in the phylogenetic trees and could be ascribed to the so-called eukaryotic 'crown' group if the composition of the latter were not so sensitive to the methods of tree building. Thus, molecular and morphological data suggest that P. palustris represents a secondarily modified eukaryotic lineage.     

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.     

Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.     

Complete modification maps for the cytosolic small and large subunit rRNAs of Euglena gracilis: functional and evolutionary implications of contrasting patterns between the two rRNA components

In the protist Euglena gracilis, the cytosolic small subunit (SSU) rRNA is a single, covalently continuous species typical of most eukaryotes; in contrast, the large subunit (LSU) rRNA is naturally fragmented, comprising 14 separate RNA molecules instead of the bipartite (28S + 5.8S) eukaryotic LSU rRNA typically seen. We present extensively revised secondary structure models of the E. gracilis SSU and LSU rRNAs and have mapped the positions of all of the modified nucleosides in these rRNAs (88 in SSU rRNA and 262 in LSU rRNA, with only 3 LSU rRNA modifications incompletely characterized). The relative proportions of ribose-methylated nucleosides and pseudouridine (∼ 60% and ∼ 35%, respectively) are closely similar in the two rRNAs; however, whereas the Euglena SSU rRNA has about the same absolute number of modifications as its human counterpart, the Euglena LSU rRNA has twice as many modifications as the corresponding human LSU rRNA. The increased levels of rRNA fragmentation and modification in E. gracilis LSU rRNA are correlated with a 3-fold increase in the level of mispairing in helical regions compared to the human LSU rRNA. In contrast, no comparable increase in mispairing is seen in helical regions of the SSU rRNA compared to its homologs in other eukaryotes. In view of the reported effects of both ribose-methylated nucleoside and pseudouridine residues on RNA structure, these correlations lead us to suggest that increased modification in the LSU rRNA may play a role in stabilizing a ‘looser’ structure promoted by elevated helical mispairing and a high degree of fragmentation.     

The systematic position of Paraspathidium Noland, 1937 (Ciliophora,Litostomatea?) inferred from primary SSU rRNA gene sequences and predicted secondary rRNA structure

Traditionally the unusual ciliate Paraspathidium has been regarded as a gymnostome haptorid (Litostomatea) based on its morphological features. In order to test this placement, the small-subunit (SSU) rRNA gene was sequenced for two isolates of Paraspathidium apofuscum and phylogenetic trees were constructed. Furthermore, the putative structure of the variable regions 2 and 4 of the SSU rRNA gene were predicted and compared with those of other ciliates. Our analyses of SSU rRNA gene sequences revealed (i) a clear separation of Paraspathidium from the haptorids and indeed the class Litostomatea, rejecting its systematic position based on morphological characters and (ii) an equally clear association with the assemblage comprising the classes Plagiopylea and Prostomatea. Putative secondary structures of the variable regions 2 and 4 of Paraspathidium are similar to those of the plagiopyleans and prostomateans but differ from the hapotrids in Helix 10, Helix E10-1 and Helix E23-5. Taken together, these results support the placement of Paraspathidium close to prostomateans and plagiopyleans, or even as a distinct group possibly at ordinal rank, within the class Plagiopylea.     

Phylogenetic analysis of the family Thermaceae with an emphasis on signature position and secondary structure of 16S rRNA

The sequences of the 16S rRNA genes from 38 strains of the family Thermaceae were compared by alignment analysis. The genus-specific and species-specific base substitutions or base deletions (signature positions) were found in three hypervariable regions (in the helices 6, 10 and 17). The differentiation of secondary structures of the high variable regions in the 5' end (38-497) containing several signature positions further supported the concept. Based on the comparisons of the secondary structures in the segments of 16S rRNAs, a key to the species of the family Thermaceae was proposed.