Sequence of the Small Subunit Ribosomal RNA Gene Expressed in the Bloodstream Stages of Plasmodium berghei: Evolutionary Implications1

ABSTRACT. We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene expressed by bloodstream stages of the apicomplexan Plasmodium berghei. It is 2059 nucleotides long. Elements contributing to its relatively large size are all concentrated in regions known to be variable in length among eukaryotes. In a phylogenetic tree constructed from pairwise comparisons of eukaryotic small subunit rRNA sequences, the apicomplexan line branches at a rather early point in eukaryotic evolution before any multicellular kingdoms had yet appeared.     

Phylogenetic position of Blastocystis hominis and of stramenopiles inferred from multiple molecular sequence data

Blastocystis hominis, a parasite of the human intestine, has recently been positioned within stramenopiles by the small subunit rRNA phylogeny. To further confirm its phylogenetic position using multiple molecular sequence data, we determined the nucleotide sequences putatively encoding small subunit ribosomal RNA, cytosolic-type 70-kDa heat shock protein, translation elongation factor 2, and the non-catalytic 'B' subunit of vacuolar ATPase of B. hominis (HE87-1 strain). Moreover, we determined the translation elongation factor 2 sequence of an apicomplexan parasite, Plasmodium falciparum, that belongs to alveolates. The maximum likelihood analyses of small subunit rRNA and cytosolic-type 70-kDa heat shock protein clearly demonstrated that B. hominis (HE87-1 strain) is positioned within stramenopiles, being congruent with the previous small subunit rRNA analysis, including the sequences of B. hominis (Nand strain) and a Blastocystis isolate from guinea pig. Although no clear resolution among major eukaryotic groups was obtained by the individual phylogenies based on the four molecules analyzed here, a combined analysis of various molecules, including these, clearly indicated that Blastocystis/stramenopiles are the closest relatives of alveolates.     

Primary and secondary structure of the 18 S ribosomal RNA of the insect species Tenebrio molitor

The sequence of the 18 S rRNA of Tenebrio molitor is reported. A detailed secondary structure model for eukaryotic small subunit rRNAs is proposed. The model comprises 48 universal helices that eukaryotic and prokaryotic small subunit rRNAs have in common, plus a number of helices in areas of variable secondary structure. For the central area of the model, an alternative structure is possible, applicable only to eukaryotic small subunit rRNAs. Possibly, small subunit rRNA switched to this alternative conformation after the eukaryotic branch had been established in evolution. Another possibility is that the two conformers represent a dynamic structural switch functioning during the translational activity of the eukaryotic ribosome.     

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.     

The nucleotide sequence of the 17S ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin

We have determined the complete sequence of the nuclear gene encoding the small subunit (17 S) rRNA of the ciliated protozoan Tetrahymena thermophila. The gene encodes an RNA molecule which is 1753 nucleotides in length. The sequence of the Tetrahymena small subunit rRNA is homologous to those of other eukaryotes, and the predicted secondary structure for the molecule includes features which are characteristic of eukaryotic small subunit rRNAs. We have also determined the nature of two different mutations in the Tetrahymena 17 S gene which result in resistance to the aminoglycoside antibiotics paromomycin and hygromycin. In each case we have identified a single base change near the 3' end of the rRNA, within a region that is highly evolutionarily conserved in both sequence and secondary structure. Analysis of the effects of these mutations on rRNA structure, and of the impact of these drugs on translation, should help to elucidate the role of the small subunit ribosomal RNA in ribosome function.     

Intermolecular base-paired interaction between complementary sequences present near the 3'' ends of 5S rRNA and 18S (16S) rRNA might be involved in the reversible association of ribosomal subunits.

Highly conserved sequences present at an identical position near the 3' ends of eukaryotic and prokaryotic 5S rRNAs are complementary to the 5' strand of the m2(6)A hairpin structure near the 3' ends of 18S rRNA and 16S rRNA, respectively. The extent of base-pairing and the calculated stabilities of the hybrids that can be constructed between 5S rRNAs and the small ribosomal subunit RNAs are greater than most, if not all, RNA-RNA interactions that have been implicated in protein synthesis. The existence of complementary sequences in 5S rRNA and small ribosomal subunit RNA, along with the previous observation that there is very efficient and selective hybridization in vitro between 5S and 18S rRNA, suggests that base-pairing between 5S rRNA in the large ribosomal subunit and 18S (16S) rRNA in the small ribosomal subunit might be involved in the reversible association of ribosomal subunits. Structural and functional evidence supporting this hypothesis is discussed.     

The troublesome parasites--molecular and morphological evidence that Apicomplexa belong to the dinoflagellate-ciliate clade.

Large insertions and deletions in the variable regions of eukaryotic 16S-like rRNA relative to the archaebacterial structure have been defined as a marker for rapidly evolving taxa. Deletions in the rRNA occur in the diplomonad Giardia and the microsporidian Vairimorpha, whereas insertions occur in Euglenozoa (Euglena and the kinetoplastids), Acanthamoeba, Naegleria, Physarum, Dictyostelium, the apicomplexan Plasmodium, the ciliate Euplotes, and some metazoa. Except Acanthamoeba and Euplotes, all of these protists were previously placed at the base of the eukaryote phylogeny. A re-analysis of the 16S-like rRNA and 5S rRNA data with the neighborliness method revealed a close relationship of Apicomplexa to the dinoflagellate-ciliate clade, most probably closer to the dinoflagellates. Morphological evidence that supports this grouping is the layer of sacs underneath the plasma membrane in all three taxa and the identical structure of trichocysts in the apicomplexan Spiromonas and dinoflagellates. The remaining rapidly evolving organisms might still be misplaced in the 16S-like rRNA trees.     

A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments

Protein synthesis in eukaryotes is mediated by both cytoplasmic and membrane-bound ribosomes. During the co-translational translocation of secretory and membrane proteins, eukaryotic ribosomes dock with the protein conducting channel of the endoplasmic reticulum. An understanding of these processes will require the detailed structure of a eukaryotic ribosome. To this end, we have compared the three-dimensional structures of yeast and rabbit ribosomes at 24 A resolution. In general, we find that the active sites for protein synthesis and translocation have been highly conserved. It is interesting that a channel was visualized in the neck of the small subunit whose entrance is formed by a deep groove. By analogy with the prokaryotic small subunit, this channel may provide a conserved portal through which mRNA is threaded into the decoding center. In addition, both the small and large subunits are built around a dense tubular network. Our analysis further suggests that the nascent chain exit tunnel and the docking surface for the endoplasmic reticulum channel are formed by this network. We surmise that many of these features correspond to rRNA, based on biochemical and structural data.Ribosomal function is critically dependent on the specific association of small and large subunits. Our analysis of eukaryotic ribosomes reveals four conserved inter-subunit bridges with a geometry similar to that found in prokaryotes. In particular, a double-bridge connects the small subunit platform with the interface canyon on the large subunit. Moreover, a novel bridge is formed between the platform and the base of the L1 domain. Finally, size differences between mammalian and yeast large subunit rRNAs have been correlated with five expansion segments that form two large spines and three extended fingers. Overall, we find that expansion segments within the large subunit rRNA have been incorporated at positions distinct from the active sites for protein synthesis and translocation.     

Secondary structure of the Dictyostelium discoideum small subunit ribosomal RNA.

We have used comparative analyses of prokaryotic and eukaryotic small subunit ribosomal RNAs to deduce a secondary structure for the Dictyostelium discoideum 18S rRNA. Most of the duplex regions are evolutionarily conserved in all organisms. We have taken advantage of the variation to the D. discoideum sequence (relative to the yeast and frog 19S rRNAs) to identify additional helical regions which are common to the eukaryotic 18S rRNAs.     

Distribution of substitution rates and location of insertion sites in the tertiary structure of ribosomal RNA

The relative substitution rate of each nucleotide site in bacterial small subunit rRNA, large subunit rRNA and 5S rRNA was calculated from sequence alignments for each molecule. Two-dimensional and three-dimensional variability maps of the rRNAs were obtained by plotting the substitution rates on secondary structure models and on the tertiary structure of the rRNAs available from X-ray diffraction results. This showed that the substitution rates are generally low near the centre of the ribosome, where the nucleotides essential for its function are situated, and that they increase towards the surface. An inventory was made of insertions characteristic of the Archaea, Bacteria and Eucarya domains, and for additional insertions present in specific eukaryotic taxa. All these insertions occur at the ribosome surface. The taxon-specific insertions seem to arise randomly in the eukaryotic evolutionary tree, without any phylogenetic relatedness between the taxa possessing them.     

Functional interaction in establishment of ribosomal integrity between small subunit protein rpS6 and translational regulator rpL10/Grc5p

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.     

Functional interaction in establishment of ribosomal integrity between small subunit protein rpS6 and translational regulator rpL10/Grc5p

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.     

Analysis of the in vivo assembly pathway of eukaryotic 40S ribosomal proteins

In eukaryotes, in vivo formation of the two ribosomal subunits from four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins (r-proteins) involves more than 150 nonribosomal proteins and around 100 small noncoding RNAs. It is temporally and spatially organized within different cellular compartments: the nucleolus, the nucleoplasm, and the cytoplasm. Here, we present a way to analyze how eukaryotic r-proteins of the small ribosomal subunit (SSU) assemble in vivo with rRNA. Our results show that key aspects of the assembly of eukaryotic r-proteins into distinct structural parts of the SSU are similar to the in vitro assembly pathway of their prokaryotic counterparts. We observe that the establishment of a stable assembly intermediate of the eukaryotic SSU body, but not of the SSU head, is closely linked to early rRNA processing events. The formation of assembly intermediates of the head controls efficient nuclear export of the SSU and cytoplasmic pre-rRNA maturation steps.     

The nucleotide sequence of the gene coding for the 16S rRNA from the archaebacterium Halobacterium halobium

The complete 1473-bp sequence of the 16S rRNA gene from the archaebacterium Halobacterium halobium has been determined. Alignment with the sequences of the 16S rRNA gene from the archaebacteria Halobacterium volcanii and Halococcus morrhua reveals similar degrees of homology, about 88%. Differences in the primary structures of H. halobium and eubacterial (Escherichia coli) 16S rRNA or eukaryotic (Dictyostelium discoideum) 18S rRNA are much higher, corresponding to 63% and 56% homology, respectively. A comparison of the nucleotide sequence of the H. halobium 16S rRNA with those of its archaebacterial counterparts generally confirms a secondary structure model of the RNA contained in the small subunit of the archaebacterial ribosome.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T(1), accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N(6)-methyladenosine-1501, and N(6),N(6)-dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N(6)-methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.     

PHYLOGENY OF GRACILARIA LEMANEIFORMIS (RHODOPHYTA) BASED ON SEQUENCE ANALYSIS OF ITS SMALL SUBUNIT RIBOSOMAL RNA CODING REGION1

The small subunit ribosomal RNA (rRNA) sequence of Gracilaria lemaneiformis Bory Weber-van Bosse was inferred from analysis of rRNA coding regions that were amplified by the polymerase chain reaction method. Comparison of the G. lemaneiformis small subunit rRNA to homologous genes of diverse eukaryotes demonstrated that the red algal divergence was nearly simultaneous with the separation of plants, fungi, animals and many other protist lineages. This result conflicts with those of 5S rRNA sequence and plastid based phytogenies which suggest that red algae represent an early divergence in the eukaryotic line of descent. Further, algae appear to be of polyphyletic origin and red algae are unrelated to higher fungi.