Application of Single-telomere length analysis (STELA), Telo-PCR and RNA-FISH to study Leishmania spp. telomere maintenance and transcription

Abstract

Telomeres are nucleoprotein structures that protect chromosome ends from fusion and degradation. Although generally considered transcriptionally silenced, it was recently demonstrated that telomeres from different eukaryotes are transcribed into long non-coding telomeric RNAs (lncRNA). In Kinetoplastida protozoa, including Leishmania, it was previously shown that different types of RNA polymerases transcribe telomeres. However, at that time, the transcription initiation site and the telomeric strand from which RNAs were transcribed was not clearly defined. TERRA (telomeric repeat-containing RNA) is an lncRNA transcribed from subtelomeric regions towards the 3´ends of telomeric repeats, whereby the C-rich telomeric strand is used as template. Several lines of evidence indicate that TERRA is essential to telomere maintenance, regulates telomere length, telomerase activity and heterochromatin deposition. Although TERRA biogenesis is well defined the functions associated to TERRA are still very controversial. We checked the expression of TERRA during the Leishmania developmental cycle using independent SL-Seq libraries constructed from the three parasite life stages (promastigotes, metacyclic and amastigotes). We validated these results using northern blot and RT-PCR. Northern blot and RT-PCR analysis confirmed the existence of TERRA transcripts comprising Leishmania specific subtelomeric sequences followed by tracts of G-rich telomeric repeats of variable length. Putative mature polyadenylated TERRA transcripts originating from some, but not all, parasite chromosome ends (right and left arms) were identified, suggesting transcription regulation at Leishmania telomeres. The expression of TERRA in Leishmania further indicates that telomere transcription is highly conserved among eukaryotes. The aim of this project is to gain expertise in the techniques STELA, Telo-PCR and RNA-FISH which will be respectively applied to study telomere length maintenance and telomere transcription in Leishmania major. We believe that they will help us to understand the role played by TERRA at Leishmania major telomeres and to study TERRA-associated functions in a pathogenic parasite. (AU)