Exploring TERRA during Leishmania major developmental cycle and continuous in vitro passages

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• Telomeres are crucial for chromosome stability and are regulated by TERRA noncoding RNAs in eukaryotes.
• TERRA is transcribed from subtelomeric regions and plays a role in chromatin regulation and telomere length maintenance.
• Trypanosomatids, including Leishmania major, possess telomeres and TERRA, but their specific functions are less understood.

Purpose of the Study:

• To investigate the expression, processing, and function of TERRA in Leishmania major.
• To determine if TERRA plays a role in telomere regulation and protection in Leishmania major.
• To explore the life-stage specific regulation of TERRA in Leishmania major.

Main Methods:

• Bioinformatic analysis to identify subtelomeric splice acceptor sites.
• SL-RNA-Seq to map splice leader sequences and confirm TERRA expression.
• Northern blotting and RT-qPCR to validate TERRA processing and abundance.
• Analysis of telomere length and TERRA-R loop formation across parasite life stages.

Main Results:

• Subtelomeric splice acceptor sites and TERRA expression were confirmed in Leishmania major.
• Leishmania major TERRA is processed via trans-splicing and polyadenylation.
• TERRA transcript abundance varied with parasite life stage, being higher in infective forms.
• No direct subtelomeric promoters for TERRA were identified; telomeric base J levels may regulate transcription.
• TERRA-R loops were detected, suggesting a role in telomere protection, particularly in infective stages.

Conclusions:

• Leishmania major TERRA shares conserved features with eukaryotic TERRA, regulating telomere length.
• TERRA exhibits life-stage specific expression and function in Leishmania major, impacting telomere protection.
• These findings reveal novel telomere-specific functions for TERRA in a significant public health parasite.