RNA synthesis in Leishmania donovani is constitutive during stage conversion: a genome-wide PRO-seq analysis

Transcription in Leishmania donovani has long been considered constitutive, as shown by nuclear run-on analyses and northern blots, with gene expression primarily regulated post-transcriptionally. Using precision nuclear run-on sequencing (PRO-seq), we investigated active transcription in axenically grown insect stages, promastigotes, and mammalian stages, amastigotes, of L. donovani. PRO-seq provides a high-resolution view of nascent RNA synthesis, allowing us to examine RNA synthesis rates and transcription initiation and termination sites genome-wide. Our results show uniform transcription across all chromosomes in all analyzed stages of the parasite's life cycle. Furthermore, we observed no significant differences in transcription levels of individual polycistronic transcription units (PTUs) between promastigotes and amastigotes during differentiation after chemical or temperature/pH induction, demonstrating that environmental conditions do not influence transcription in L. donovani. Additionally, transcription was confirmed to be strand-specific within PTUs, with initiation and termination occurring at strand switch regions. We successfully identified sites of transcription initiation and termination, which can be used for future integration with epigenetic data. These findings reinforce that transcription in L. donovani is constitutive and stage-independent, excluding a role in gene expression regulation.

Our data unequivocally show that life cycle stage-dependent gene expression in Leishmania donovani, the parasite responsible for the lethal Kala-Azar disease, does not involve regulated RNA synthesis. We used a genome-wide analysis of RNA synthesis of cultivated parasites of three different differentiation forms to measure nascent RNA synthesis and found no significant changes. Together with earlier data, this implies mechanisms of gene expression control that set Leishmania apart from its human and animal hosts.