Multigene-based analyses on evolutionary phylogeny of two controversial ciliate orders: Pleuronematida and Loxocephalida (Protista, Ciliophora, Oligohymenophorea).

Relationships among members of the ciliate subclass Scuticociliatia (Ciliophora, Oligohymenophorea) are largely unresolved. Phylogenetic studies of its orders Pleuronematida and Loxocephalida were initially based on small subunit ribosomal RNA gene (SSU-rDNA) analyses of a limited number of taxa. Here we characterized 37 sequences (SSU-rDNA, ITS-5.8S and LSU-rDNA) from 21 taxonomically controversial members of these orders. Phylogenetic trees constructed to assess the inter- and intra-generic relationships of pleuronematids and loxocephalids reveal the following: (1) the order Loxocephalida and its two families Loxocephalidae and Cinetochilidae are not monophyletic when more taxa are added; (2) the core pleuronematids are divided into two fully supported clades, however, the order Pleuronematida is not monophyletic because Cyclidium glaucoma is closer to Thigmotrichida; (3) the family Pleuronematidae and the genus Schizocalyptra are monophyletic, though rDNA sequences of Pleuronema species are highly variable; (4) Pseudoplatynematum and Sathrophilus are closely related to the subclass Astomatia, while Cinetochilum forms a monophyletic group with the subclass Apostomatia; and (5) Hippocomos falls in the order Pleuronematida and is closely related to Eurystomatellidae and Cyclidium plouneouri. Further, in an effort to provide a better resolution of evolutionary relationships, the secondary structures of ITS2 transcripts and the variable region 4 (V4) of the small subunit ribosomal RNA (SSU-rRNA) are predicted, revealing that ITS2 structures are conserved at the order level while V4 region structures are more variable than ITS2 structures.

Experimental identification and analysis of macronuclear non-coding RNAs from the ciliate Tetrahymena thermophila.

The ciliate Tetrahymena thermophila is an important eukaryotic model organism that has been used in pioneering studies of general phenomena, such as ribozymes, telomeres, chromatin structure and genome reorganization. Recent work has shown that Tetrahymena has many classes of small RNA molecules expressed during vegetative growth or sexual reorganization. In order to get an overview of medium-sized (40-500 nt) RNAs expressed from the Tetrahymena genome, we created a size-fractionated cDNA library from macronuclear RNA and analyzed 80 RNAs, most of which were previously unknown. The most abundant class was small nucleolar RNAs (snoRNAs), many of which are formed by an unusual maturation pathway. The modifications guided by the snoRNAs were analyzed bioinformatically and experimentally and many Tetrahymena-specific modifications were found, including several in an essential, but not conserved domain of ribosomal RNA. Of particular interest, we detected two methylations in the 5'-end of U6 small nuclear RNA (snRNA) that has an unusual structure in Tetrahymena. Further, we found a candidate for the first U8 outside metazoans, and an unusual U14 candidate. In addition, a number of candidates for new non-coding RNAs were characterized by expression analysis at different growth conditions.

Stress-induced tRNA-derived RNAs: a novel class of small RNAs in the primitive eukaryote Giardia lamblia.

Giardia lamblia is an early diverging and evolutionarily successful protozoan as it can enter into a dormant cyst stage from a vegetative trophozoite. During dormant stage, its metabolic rate decreases dramatically. However, to date, the regulatory molecules participating in the initiation and maintenance of this process have not been fully investigated. In this study, we have identified a class of abundant small RNAs named sitRNAs, which are approximately 46 nucleotides in length and accumulate in G. lamblia encysting cultures. Remarkably, they are derived from the 3' portion of fully matured tRNAs by cleavage of the anticodon left arm, with the 3' terminal CCA triplex still connected. During differentiation, only a limited portion of mature tRNAs is cleaved, but this cleavage occurs almost in the entire tRNA family. sitRNAs begin to accumulate as early as 3 h after initiation of encystation and are maintained at a relatively stable level during the whole process, exhibiting an expression peak at around 24 hr. Our studies further show that sitRNAs can be induced by several other stress factors, and in the case of serum deprivation, both tRNAs and sitRNAs degrade rapidly, with the accumulation of tRNA being halved. Our results may provide new insight into a novel mechanism for stressed G. lamblia to regulate gene expression globally.

Identification of novel guide RNAs from the mitochondria of Trypanosoma brucei.

The majority of mitochondrial mRNAs in African trypanosomes are subject to an RNA editing reaction, which is characterized by the insertion and/or deletion of U nucleotides only. The reaction creates functional mRNAs and is catalyzed by a high molecular mass enzyme complex, the editosome. Editosomes interact with a unique class of small non-coding, 3'-oligouridylated (oU) RNAs, so-called guide RNAs (gRNAs). Guide RNAs function as transacting templates in the U deletion/insertion reaction and thus, represent key components in the reaction cycle. Furthermore, by utilizing different gRNAs, alternative editing events can take place, thereby expanding the protein diversity in the mitochondria of the parasites. In this study, we have analyzed small, non-coding mitochondrial transcripts from Trypanosoma brucei. By generating cDNA libraries from size-selected RNA populations we identified 51 novel oU-RNAs. For 29 of these RNAs we were able to predict cognate mRNA targets. By Northern blot analysis, we verified the expression of 22 of these oU-RNAs and demonstrate that they share all known gRNA characteristics. Five of these 51 putative gRNAs are characterized by single mismatches to their cognate, fully edited mRNA sequences suggesting that they could act as gRNAs for alternative editing events.

No miRNA were found in Plasmodium and the ones identified in erythrocytes could not be correlated with infection.

BACKGROUND: The transcriptional regulation of Plasmodium during its complex life cycle requires sequential activation and/or repression of different genetic programmes. MicroRNAs (miRNAs) are a highly conserved class of non-coding RNAs that are important in regulating diverse cellular functions by sequence-specific inhibition of gene expression. What is know about double-stranded RNA-mediated gene silencing (RNAi) and posttranscriptional gene silencing (PTGS) in Plasmodium parasites entice us to speculate whether miRNAs can also function in Plasmodium-infected RBCs. RESULTS: Of 132 small RNA sequences, no Plasmodium-specific miRNAs have been found. However, a human miRNA, miR-451, was highly expressed, comprising approximately one third of the total identified miRNAs. Further analysis of miR-451 expression and malaria infection showed no association between the accumulation of miR-451 in Plasmodium falciparum-iRBCs, the life cycle stage of P. falciparum in the erythrocyte, or of P. berghei in mice. Moreover, treatment with an antisense oligonucleotide to miR-451 had no significant effect on the growth of the erythrocytic-stage P. falciparum. METHODS: Short RNAs from a mixed-stage of P. falciparum-iRBC were separated in a denaturing polyacrylamide gel and cloned into T vectors to create a cDNA library. Individual clones were then sequenced and further analysed by bioinformatics prediction to discover probable miRNAs in P. falciparum-iRBC. The association between miR-451 expression and the parasite were analysed by Northern blotting and antisense oligonucleotide (ASO) of miR-451. CONCLUSION: These results contribute to eliminate the probability of miRNAs in P. falciparum. The absence of miRNA in P. falciparum could be correlated with absence of argonaute/dicer genes. In addition, the miR-451 accumulation in Plasmodium-infected RBCs is independent of parasite infection. Its accumulation might be only the residual of erythroid differentiation or a component to maintain the normal function of mature RBCs.

Pseudoknot structures with conserved base triples in telomerase RNAs of ciliates.

Telomerase maintains the integrity of telomeres, the ends of linear chromosomes, by adding G-rich repeats to their 3'-ends. Telomerase RNA is an integral component of telomerase. It contains a template for the synthesis of the telomeric repeats by the telomerase reverse transcriptase. Although telomerase RNAs of different organisms are very diverse in their sequences, a functional non-template element, a pseudoknot, was predicted in all of them. Pseudoknot elements in human and the budding yeast Kluyveromyces lactis telomerase RNAs contain unusual triple-helical segments with AUU base triples, which are critical for telomerase function. Such base triples in ciliates have not been previously reported. We analyzed the pseudoknot sequences in 28 ciliate species and classified them in six different groups based on the lengths of the stems and loops composing the pseudoknot. Using miniCarlo, a helical parameter-based modeling program, we calculated 3D models for a representative of each morphological group. In all cases, the predicted structure contains at least one AUU base triple in stem 2, except for that of Colpidium colpoda, which contains unconventional GCG and AUA triples. These results suggest that base triples in a pseudoknot element are a conserved feature of all telomerases.

Discovery and barcoding by analysis of spliced leader RNA gene sequences of new isolates of Trypanosomatidae from Heteroptera in Costa Rica and Ecuador.

Trypanosomatid diversity in Heteroptera was sampled using a culture-independent approach based on amplification and sequencing of Spliced Leader RNA gene repeats from environmental samples. By combining the data collected herein with that of previous work, the prevalence of parasites was found to be 22%-23%. Out of approximately 170 host species investigated nearly 60 were found to harbor trypanosomatids. The parasites found were grouped by cluster analysis into 48 typing units. Most of these were well separated from the known groups and, therefore, likely represent new trypanosomatid species. The sequences for each typing unit serve as barcodes to facilitate their recognition in the future. As the sampled host species represent a minor fraction of potential hosts, the entire trypanosomatid diversity is far greater than described thus far. Investigations of trypanosomatid diversity, host-specificity, and biogeography have become feasible using the approach described herein.

Two classes of endogenous small RNAs in Tetrahymena thermophila.

Endogenous small RNAs function in RNA interference (RNAi) pathways to guide RNA cleavage, translational repression, or methylation of DNA or chromatin. In Tetrahymena thermophila, developmentally regulated DNA elimination is governed by an RNAi mechanism involving approximately 27-30-nucleotide (nt) RNAs. Here we characterize the sequence features of the approximately 27-30-nt RNAs and a approximately 23-24-nt RNA class representing a second RNAi pathway. The approximately 23-24-nt RNAs accumulate strain-specifically manner and map to the genome in clusters that are antisense to predicted genes. These findings reveal the existence of distinct endogenous RNAi pathways in the unicellular T. thermophila, a complexity previously demonstrated only in multicellular organisms.

Ribosomal RNA phylogeny of bodonid and diplonemid flagellates and the evolution of euglenozoa.

Euglenozoa is a major phylum of excavate protozoa (comprising euglenoids, kinetoplastids, and diplonemids) with highly unusual nuclear, mitochondrial, and chloroplast genomes. To improve understanding of euglenozoan evolution, we sequenced nuclear small-subunit rRNA genes from 34 bodonids (Bodo, Neobodo, Parabodo, Dimastigella-like, Rhynchobodo, Rhynchomonas, and unidentified strains), nine diplonemids (Diplonema, Rhynchopus), and a euglenoid (Entosiphon). Phylogenetic analysis reveals that diplonemids and bodonids are more diverse than previously recognised, but does not clearly establish the branching order of kinetoplastids, euglenoids, and diplonemids. Rhynchopus is holophyletic; parasitic species arose from within free-living species. Kinetoplastea (bodonids and trypanosomatids) are robustly holophyletic and comprise a major clade including all trypanosomatids and most bodonids ('core bodonids') and a very divergent minor one including Ichthyobodo. The root of the major kinetoplastid clade is probably between trypanosomatids and core bodonids. Core bodonids have three distinct subclades. Clade 1 has two distinct Rhynchobodo-like lineages; a lineage comprising Dimastigella and Rhynchomonas; and another including Cruzella and Neobodo. Clade 2 comprises Cryptobia/ Trypanoplasma, Procryptobia, and Parabodo. Clade 3 is an extensive Bodo saltans species complex. Neobodo designis is a vast genetically divergent species complex with mutually exclusive marine and freshwater subclades. Our analysis supports three phagotrophic euglenoid orders: Petalomonadida (holophyletic), Ploeotiida (probably holophyletic), Peranemida (paraphyletic).

Phylogeny of choanozoa, apusozoa, and other protozoa and early eukaryote megaevolution.

The primary diversification of eukaryotes involved protozoa, especially zooflagellates-flagellate protozoa without plastids. Understanding the origins of the higher eukaryotic kingdoms (two purely heterotrophic, Animalia and Fungi, and two primarily photosynthetic, Plantae and Chromista) depends on clarifying evolutionary relationships among the phyla of the ancestral kingdom Protozoa. We therefore sequenced 18S rRNA genes from 10 strains from the protozoan phyla Choanozoa and Apusozoa. Eukaryote diversity is encompassed by three early-radiating, arguably monophyletic groups: Amoebozoa, opisthokonts, and bikonts. Our taxon-rich rRNA phylogeny for eukaryotes allowing for intersite rate variation strongly supports the opisthokont clade (animals, Choanozoa, Fungi). It agrees with the view that Choanozoa are sisters of or ancestral to animals and reveals a novel nonflagellate choanozoan lineage, Ministeriida, sister either to choanoflagellates, traditionally considered animal ancestors, or to animals. Maximum likelihood trees suggest that within animals Placozoa are derived from medusozoan Cnidaria (we therefore place Placozoa as a class within subphylum Medusozoa of the Cnidaria) and hexactinellid sponges evolved from demosponges. The bikont and amoebozoan radiations are both very ill resolved. Bikonts comprise the kingdoms Plantae and Chromista and three major protozoan groups: alveolates, excavates, and Rhizaria. Our analysis weakly suggests that Apusozoa, represented by Ancyromonas and the apusomonads ( Apusomonas and the highly diverse and much more ancient genus Amastigomonas, from which it evolved), are not closely related to other Rhizaria and may be the most divergent bikont lineages. Although Ancyromonas and apusomonads appear deeply divergent in 18S rRNA trees, the trees neither refute nor support the monophyly of Apusozoa. The bikont phylum Cercozoa weakly but consistently appears as sister to Retaria (Foraminifera; Radiolaria), together forming a hitherto largely unrecognized major protozoan assemblage (core Rhizaria) in the eukaryote tree. Both 18S rRNA sequence trees and a rare deletion show that nonciliate haplosporidian and paramyxid parasites of shellfish (together comprising the Ascetosporea) are not two separate phyla, as often thought, but part of the Cercozoa, and may be related to the plant-parasitic plasmodiophorids and phagomyxids, which were originally the only parasites included in the Cercozoa. We discuss rRNA trees in relation to other evidence concerning the basal diversification and root of the eukaryotic tree and argue that bikonts and opisthokonts, at least, are holophyletic. Amoebozoa and bikonts may be sisters-jointly called anterokonts, as they ancestrally had an anterior cilium, not a posterior one like opisthokonts; this contrasting ciliary orientation may reflect a primary divergence in feeding mode of the first eukaryotes. Anterokonts also differ from opisthokonts in sterol biosynthesis (cycloartenol versus lanosterol pathway), major exoskeletal polymers (cellulose versus chitin), and mitochondrial cristae (ancestrally tubular not flat), possibly also primary divergences.

Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhadtii.

Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs, and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from reverse transcriptase-polymerase chain reaction protocols used to select polyadenylated RNAs revealed that, at least for the mRNAs and tRNAs, there are three apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3' end, even when this follows a strong secondary structure. In the second case, the tail is added to pre-mRNA or pre-tRNA, suggesting a possible competition between polyadenylation and RNA-processing pathways. Finally, in all cases, the poly(A) tail can be added internally, possibly as a part of an RNA-decay pathway. The tails found in Chlamydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly between 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein extract showed that a 3' end A25 tail was sufficient to stimulate rapid degradation of atpB RNA in vitro, with a lesser effect for petD, and only minor effects on trnE. We therefore propose that polyadenylation contributes to mRNA degradation in Chlamydomonas chloroplasts, but that its effect may vary.