Telomerase in kinetoplastid parasitic protozoa

Abstract

We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species: Trypanosoma brucei, Leishmania major, and Leishmania tarentolae. Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N., et al. The activity in T. brucei extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of T. brucei. Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.Chromosomal termini, or telomeres, in most eukaryotes consist of DNA-protein complexes that are essential for genomic integrity and cell viability. Part of the telomeric DNA that is lost during each round of cell replication is replaced primarily by the action of a ribonucleoprotein enzyme complex telomerase (1). Telomerase activity, first described in the ciliated protozoan Tetrahymena thermophila (1), is widely distributed among phylogenetically diverse eukaryotes including yeasts, amphibians, mammals, and plants, and was recently reported in the pathogenic malaria parasite Plasmodium falciparum (2-8). Telomerase adds dNTPs to the 3Ј end of the G-rich strand of chromosomal DNA by reverse transcription of a telomeric sequence template within the telomerase RNA subunit. Mutations in the template region of the telomerase RNA gene cause progressive shortening of the telomeres and cell senescence in T. thermophila (9). In mice, knock-out of telomerase RNA leads to increased telomere shortening, chromosomal aberrations, and sterility (10). The telomerase RNA subunit is generally difficult to identify because there is only limited sequence conservation at the level of secondary structure even between related species (ref. 11, and reviewed in ref. 12), necessitating more traditional biochemical approaches for its identification.The cloning and characterization of the protein catalytic subunit (telomerase reverse transcriptase, TERT) of Euplotes aediculatus and Saccharomyces cerevisiae telomerases by Lingner et al. (13) made possible the identification of telomerase reverse transcriptase homologues in Schizosaccharomyces pombe and humans (14) and the ciliates T. thermophila and Oxytricha trifallax (15,16). The amino acid sequences of these homologues indicate that the telomerase protein subunit is a specialized reverse transcriptase, evolutionarily most related to reverse transcriptases in the non-long terminal repeat retrotransposon family (ref.

A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei

“…In TbTR-DKO cells expressing TbTRt mutant, most of the mutant telomeres only had a short stretch of mutant sequence incorporated, suggesting low processivity of T. brucei telomerase. This is consistent with our telomerase primer extension result and previous observations [28].…”

“…Therefore, altering the TbTR template sequence can have a profound effect on the telomere structure, which may have therapeutic implications. In addition, previous studies proposed a putative template domain for Trypanosoma telomerase RNA [28,38], which has a unique cytosine-rich motif distinctive from any other known TR molecules, except that from another pathogenic protozoa Plasmodium falciparum (Supplementary information, Figure S1A). Through mutation analysis, we confirmed that this motif indeed is part of TbTR template.…”

“…Trypanosome telomerase is semiprocessive in nature [28,38], although it makes telomeric repeats identical to those synthesized by human telomerase, a highly processive enzyme. Such difference in enzyme processivity can be due to the differences in core template sequences or template adjacent region between host and pathogen.…”

“…Based on the major telomeric repeat sequence (TTAGGG) identified and considering the previously suggested TR template sequence [28,38] (Supplementary information, Figure S1A), we performed a BLAST search for short, nearly exact matching sequences in the T. brucei genome. In this process we identified a number of initial TR candidates (Supplementary information, Figure S1B), many of which overlapped with annotated ORFs, structural RNAs (rRNAs, tRNAs or snoRNAs) and sequences at the telomeric ends of T. brucei genome (Supplementary information, Figure S1B-i).…”

Telomere shortening and cell cycle arrest in Trypanosoma brucei expressing human telomeric repeat factor TRF1

“…Transcriptionally active telomeres grow slightly faster (10 bp per cell division), suggesting a correlation between telomere structure and the transcriptional status of the genes located in their proximity [20]. Telomerase activity with limited processivity has been described in three species of kinetoplastids, including T. brucei [21]. In addition, position effects within the ES [22][23][24], and chromatin remodeling and developmentally regulated repression effects close to telomeres [16], suggest a role for telomeres in regulating antigenic variation and suggest that there are differences in telomeric chromatin structure between BF and PF trypanosomes.…”