The UTRs of Leishmania donovani vary in length and are enriched in potential regulatory structures
Leishmania spp. regulate gene expression largely post-transcriptionally, yet untranslated regions (UTRs) remain poorly delineated. We generated high-quality genome and transcriptome datasets for Leishmania donovani strain 1S2D (Ld1S) by combining PacBio HiFi de novo assembly with Oxford Nanopore direct RNA sequencing of promastigotes and axenic amastigotes. The genome assembly consists of 65 scaffolds totaling [~]33.3 Mb. Structural comparisons to LdBPK282A1 revealed numerous rearrangements, including some reshufling genes among polycistronic transcription units and validated by polycistronic reads from RNA sequencing. 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 UTRs. We also noted that 31.9% and 11.5% were expressed into multiple isoforms in promastigotes and amastigotes, respectively. Collectively, these data provide a comprehensive 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 UTR in post-transcriptional regulation in L. donovani. Author SummaryLeishmania donovani parasites cause visceral leishmaniasis, a deadly disease affecting hundreds of thousands of people worldwide. Unlike most eukaryotes, these parasites do not regulate their genes mainly at the level of transcription. Instead, control happens after the genes are transcribed, and much of this regulation depends on regions of RNA that are not translated into protein, called untranslated regions (UTRs). However, UTRs in Leishmania have remained poorly characterized. Here we generated high-quality genome and transcriptome resources for a clinical strain of L. donovani. By combining state-of-the-art long-read sequencing technologies, we precisely mapped thousands of UTRs and discovered that many genes produce transcripts with variable UTR lengths that differ between parasite life stages. We also found that UTRs are 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 central role in controlling how genes are expressed during the parasites life cycle. Our work lays a foundation for future studies aimed at understanding parasite biology and identifying new targets for intervention.