# The UTRs of Leishmania donovani vary in length and are enriched in potential regulatory structures

## TL;DR

This study provides a detailed analysis of untranslated regions in Leishmania donovani, revealing their role in gene regulation and structural variations.

## Contribution

The paper presents the first comprehensive genome-wide annotation of UTRs in Leishmania donovani using advanced sequencing technologies.

## Key findings

- UTRs in Leishmania donovani are enriched in RNA G-quadruplex structures, suggesting a role in post-transcriptional regulation.
- Significant stage-specific UTR length polymorphisms were observed in promastigotes and amastigotes.
- The study identified thousands of UTRs and validated gene expression patterns across life stages.

## Abstract

Leishmania spp. regulate gene expression posttranscriptionally, yet untranslated regions (UTRs) that can affect mRNA stability and translation remain poorly delineated. We generated a de novo assembled genome for Leishmania donovani strain 1S2D (Ld1S) using PacBio HiFi and characterized the transcriptomes of promastigotes and axenic amastigotes with Oxford Nanopore direct RNA sequencing. The genome assembly consists of 65 scaffolds totaling ~33.3 Mb. Structural comparisons to LdBPK282A1 revealed numerous rearrangements, including genes reshuffled among polycistronic transcription units and validated by RNA sequencing of polycistronic reads. Promastigote and amastigote RNA sequencing produced 469,010 and 46,729 monocistronic reads containing a spliced-leader and a polyA tail sequences, defining 8,479 transcripts and supporting 7,415 of the 7,969 annotated protein-coding genes, as well as 604 putative long non-coding RNAs. We annotated UTRs for 4,921 genes and observed that putative RNA G-quadruplexes were markedly enriched in these regions. We also noted that 31.9% and 11.5% of genes were expressed into multiple isoforms in promastigotes and amastigotes, respectively. Collectively, these data provide a genome-wide annotation of L. donovani genes and their UTRs and reveal widespread and stage-specific UTR length polymorphisms and, overall, points to an important role of 3’ UTRs in post-transcriptional regulation in L. donovani.

Leishmania donovani parasites cause visceral leishmaniasis, a deadly disease affecting hundreds of thousands of people worldwide. Unlike most eukaryotes, Leishmania parasites do not regulate their genes at the level of transcription. Instead, gene regulation happens after the genes are transcribed, and much of this regulation likely depends on the regions of the mRNAs that are not translated into protein, the untranslated regions (UTRs). However, UTRs in Leishmania remained poorly characterized. Here, we generated high-quality genome and transcriptome data for a strain of L. donovani commonly used in laboratory experiments. By combining state-of-the-art long-read sequencing technologies, we precisely annotated thousands of UTRs and discovered that many genes produced transcripts with variable UTR lengths. We also observed that UTRs were enriched in RNA structures called G-quadruplexes, which are known to influence gene regulation. These findings provide the most comprehensive view to date of UTRs in Leishmania and highlight their likely role in controlling how genes are expressed during the parasite’s life cycle. Our work lays a foundation for future studies aiming at better understanding parasite biology and identifying new targets for intervention.