Regulation and trafficking of three distinct 18 S ribosomal RNAs during development of the malaria parasite.

The human malaria parasite Plasmodium vivax has been shown to regulate the transcription of two distinct 18 RNAs during development. Here we show a third and distinctive type of ribosome that is present shortly after zygote formation, a transcriptional pattern of ribosome types that relates closely to the developmental state of the parasite and a phenomenon that separates ribosomal types at a critical phase of maturation. The A-type ribosome is predominantly found in infected erythrocytes of the vertebrate and the mosquito blood meal. Transcripts from the A gene are replaced by transcripts from another locus, the O gene, shortly after fertilization and increase in number as the parasite develops on the mosquito midgut. Transcripts from another locus, the S gene, begins as the oocyst form of the parasite matures. RNA transcripts from the S gene are preferentially included in sporozoites that bud off from the oocyst and migrate to the salivary gland while the O gene transcripts are left within the oocyst. Although all three genes are typically eukaryotic in structure, the O gene transcript, described here, varies from the other two in core regions of the rRNA that are involved in mRNA decoding and translational termination. We now can correlate developmental progression of the parasite with changes in regions of rRNA sequence that are broadly conserved, where sequence alterations have been related to function in other systems and whose effects can be studied outside of Plasmodium. This should allow assessment of the role of translational control in parasite development.

Nuclear-encoded rDNA group I introns: origin and phylogenetic relationships of insertion site lineages in the green algae.

Group I introns are widespread in eukaryotic organelles and nuclear-encoded ribosomal DNAs (rDNAs). The green algae are particularly rich in rDNA group I introns. To better understand the origins and phylogenetic relationships of green algal nuclear-encoded small subunit rDNA group I introns, a secondary structure-based alignment was constructed with available intron sequences and 11 new subgroup ICI and three new subgroup IB3 intron sequences determined from members of the Trebouxiophyceae (common phycobiont components of lichen) and the Ulvophyceae. Phylogenetic analyses using a weighted maximum-parsimony method showed that most group I introns form distinct lineages defined by insertion sites within the SSU rDNA. The comparison of topologies defining the phylogenetic relationships of 12 members of the 1512 group I intron insertion site lineage (position relative to the E. coli SSU rDNA coding region) with that of the host cells (i.e., SSU rDNAs) that contain these introns provided insights into the possible origin, stability, loss, and lateral transfer of ICI group I introns. The phylogenetic data were consistent with a viral origin of the 1512 group I intron in the green algae. This intron appears to have originated, minimally, within the SSU rDNA of the common ancestor of the trebouxiophytes and has subsequently been vertically inherited within this algal lineage with loss of the intron in some taxa. The phylogenetic analyses also suggested that the 1512 intron was laterally transferred among later-diverging trebouxiophytes; these algal taxa may have coexisted in a developing lichen thallus, thus facilitating cell-to-cell contact and the lateral transfer. Comparison of available group I intron sequences from the nuclear-encoded SSU rDNA of phycobiont and mycobiont components of lichens demonstrated that these sequences have independent origins and are not the result of lateral transfer from one component to the other.

Comprehensive comparison of structural characteristics in eukaryotic cytoplasmic large subunit (23 S-like) ribosomal RNA.

Comparative modeling of secondary structure is a proven approach to predicting higher order structural elements in homologous RNA molecules. Here we present the results of a comprehensive comparison of newly modeled or refined secondary structures for the cytoplasmic large subunit (23 S-like) rRNA of eukaryotes. This analysis, which covers a broad phylogenetic spectrum within the eukaryotic lineage, has defined regions that differ widely in their degree of structural conservation, ranging from a core of primary sequence and secondary structure that is virtually invariant, to highly variable regions. New comparative information allows us to propose structures for many of the variable regions that had not been modeled before, and rigorously to confirm or refine variable region structures previously proposed by us or others. The present analysis also serves to identify phylogenetically informative features of primary and secondary structure that characterize these models of eukaryotic cytoplasmic 23 S-like rRNA. Finally, the work summarized here provides a basis for experimental studies designed both to test further the validity of the proposed secondary structures and to explore structure-function relationships.

Primary and secondary structure analyses of the rDNA group-I introns of the Zygnematales (Charophyta).

The Zygnematales (Charophyta) contain a group-I intron (subgroupIC1) within their nuclear-encoded small subunit ribosomal DNA (SSU rDNA) coding region. This intron, which is inserted after position 1506 (relative to the SSU rDNA of Escherichia coli), is proposed to have been vertically inherited since the origin of the Zygnematales approximately 350-400 million years ago. Primary and secondary structure analyses were carried out to model group-I intron evolution in the Zygnematales. Secondary structure analyses support genetic data regarding sequence conservation within regions known to be functionally important for in vitro self-splicing of group-I introns. Comparisons of zygnematalean group-I intron secondary structures also provided some new insights into sequences that may have important roles in in vivo RNA splicing. Sequence analyses showed that sequence divergence rates and the nucleotide compositions of introns and coding regions within any one taxon varied widely, suggesting that the "1506" group-I introns and rDNA coding regions in the Zygnematales evolve independently.

Structural features of the large subunit rRNA expressed in Plasmodium falciparum sporozoites that distinguish it from the asexually expressed subunit rRNA.

The developmentally regulated transcription of at least two distinct sets of nuclear-encoded ribosomal RNAs is detected in Plasmodium species. The identification of functional differences between the two sets of rRNAs is of interest. To facilitate the search for such differences, we have identified the 5.8S and 28S rRNAs from Plasmodium falciparum that are expressed in the sporozoite stage (S gene) of the parasites' life cycle in the mosquito host and compare them to transcripts expressed in the red blood cells (A gene) of the vertebrate host. This completes the first set of A- and S-type nuclear-encoded rRNA genes for a Plasmodium species. Analysis of the predicted secondary structures of the two units reveals the majority of differences between the A- and S-type genes occur in regions previously known to be variable. However, the predicted secondary structure of both 28S rRNAs indicates 11 positions within conserved areas that are not typical of eucaryotic rRNAs. Although the A-type gene resembles almost all eucaryotes, being atypical in only 4 of the 11 positions, the S gene is variant in 8 of the 11 positions. In three of these positions, the S-type gene resembles the consensus nucleotides for the 23S rRNA from Eubacteria and/or Archaea. A few differences occur in regions associated with ribosome function, in particular the GTPase site where the S-type differs in a base pair and loop from all known sequences. Further, the identification of compensatory changes at conserved points of interactions between the 5.8S-28S rRNAs indicates that transcripts from A- and S-units should not be interchangeable.

Identification of base-triples in RNA using comparative sequence analysis.

Comparative sequence analysis has proven to be a very efficient tool for the determination of RNA secondary structure and certain tertiary interactions. However, base-triples, an important RNA structural element, cannot be predicted accurately from sequence data. We show here that the poor base correlations observed at base-triple positions are the result of two factors. (1) Base covariation is not as strictly required in triples as it is in Watson-Crick pairs. (2) Base-triple structures are less conserved among homologous molecules. A particularity of known triple-helical regions is the presence of multiple base correlations that do not reflect direct pairing. We suggest that natural mutations in base-triples create structural changes that require compensatory mutations in adjacent base-pairs and triples to maintain the triple-helix conformation. On the basis of these observations, we devised two new measures of association that significantly enhance the base-triple signal in correlation studies. We evaluated correlations between base-pairs and single stranded bases, and correlations between adjacent base-pairs. Positions that score well in both analyses are the best triple candidates. This procedure correctly identifies triples, or interactions very close to the proposed triples, in type I and type II tRNAs and in the group I intron.

Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years.

A comparative database of group I intron structures.

We have created a database of comparatively derived group I intron secondary structure diagrams. This collection currently contains a broad sampling of phylogenetically and structurally similar and diverse structures from over 200 publicly available intron sequences. As more group I introns are sequenced and added to the database, we anticipate minor refinements in these secondary structure diagrams. These diagrams are directly accessible by computer as well as from the authors.

Representation of the secondary and tertiary structure of group I introns.

Group I introns, which are widespread in nature, carry out RNA self-splicing. The secondary structure common to these introns was for the most part established a decade ago. Information about their higher order structure has been derived from a range of experimental approaches, comparative sequence analysis, and molecular modelling. This information now provides the basis for a new two-dimensional structural diagram that more accurately represents the domain organization and orientation of helices within the intron, the coaxial stacking of certain helices, and the proximity of key nucleotides in three-dimensional space. It is hoped that this format will facilitate the detailed comparison of group I intron structures.