Frataxin, a conserved mitochondrial protein, in the hydrogenosome of Trichomonas vaginalis

Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue (T. vaginalis frataxin) from the human parasite Trichomonas vaginalis. Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical alpha-beta sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis. When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes.     

The ATP-binding cassette proteins of the deep-branching protozoan parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters.     

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

A 25-bp ancient spliceosomal intron in the TvRab1a gene of Trichomonas vaginalis

Spliceosomal introns play a key role in eukaryotic genome evolution and protein diversity. A large Rab GTPase family has been identified in a unicellular eukaryote Trichomonas vaginalis. However, the characteristics of introns in Rab genes of T. vaginalis have not been investigated previously. In this study, we identified a 25-bp spliceosomal intron in the T. vaginalis Rab1a (TvRab1a) gene, the smallest intron in T. vaginalis to be characterized to date. This intron contains a canonical splice site at both 5' (GT) and 3' (AG) ends, and a putative branch-point sequence (TCTAAC) that matches the Trichomonad consensus sequence of ACTAAC except for the first nucleotide. The position and phase of the TvRab1a intron are evolutionarily conserved in Rab1 homologous genes across at least five eukaryotic supergroups, including Opisthokonta, Amoebozoa, Excavata, Chromalveolata, and Plantae. These results strongly suggest that the TvRab1a intron is likely to be an ancient spliceosomal intron, and it can therefore be used as a phylogenetic marker to evaluate particular eukaryotic groupings. Identification and characterization of the TvRabla intron may provide an insight into the evolution of the large Rab repertoire in T. vaginalis.     

Linked Genes for Calmodulin and E2 Ubiquitin-Conjugating Enzyme in Trichomonas vaginalis

ABSTRACT In searching the genomes of early-diverging protists to study whether the possession of calmodulin is ancestral to all eukaryotes, the gene for calmodulin was identified in Trichomonas vaginalis. This flagellate is a member of the Parabasalia, one of the earliest lineages of recognized eukaryotes to have diverged. This sequence was used to isolate a homologous 1.250-kb fragment from the T. vaginalis genome by inverse polymerase chain reaction. This fragment was also completely sequenced and shown to contain the 3' end of the single-copy calmodulin gene and the 3' end of a gene encoding a protein with high similarity to E2 ubiquitin-conjugating enzymes, a family which has previously only been identified in animals, plants, and fungi. Phylogenetic analysis of 50 members of the E2 family distinguishes at least nine separate subfamilies one of which includes the T. vaginalis E2-homologue and an uncharacterized gene from yeast chromosome XII.