Novel insights into the Leishmania infantum transcriptome diversity of protein-coding and non-coding sequences in both stages of parasite development using nanopore direct RNA sequencing.

Background: relies on posttranscriptional control to regulate gene expression. Protein-coding genes are synthesised as polycistronic precursors that are processed into individual mRNAs by -splicing adding the spliced leader (SL) RNA to the 5’-end and 3’ cleavage-polyadenylation. Here, we employ Nanopore direct RNA sequencing (DRS) combined with Illumina RNA-Seq to comprehensively interrogate the transcriptomes of developmental stages at single-molecule resolution.

Results: Analysis of DRS full-length reads of poly(A)+-enriched RNA from developmental stages enabled us to precisely determine the primary SL and poly(A) sites for 52% of the protein-coding transcripts and to accurately define their 5’- and 3’-end and the length of UTRs. In addition, our analysis confirmed the motifs ‘[C/A/T] A|G’ being associated with 94.8% of the SL cleavage sites and better defined the genomic context for cleavage and polyadenylation. Overall, we observed more diversity for poly(A) than SL sites per transcript. The frequency of the primary SL and poly(A) sites was 64.2% and 24%, respectively, with most transcripts having additional poly(A) sites nearby. Alternative polyadenylation was detected in 11-13% of transcripts with ~ 20% of these having different primary poly(A) sites between promastigote and amastigote developmental stages. Furthermore, DRS uncovered multiple processing events occurring mostly within 3’UTRs, leading to the formation of long non-coding RNAs (lncRNAs). The transcriptome expresses a rich repertoire of 1,825 lncRNAs, of which 98% were not previously annotated in and only 21.5% were found in . These lncRNAs exhibit generally distinct expression patterns from the 3’UTRs they derived and several are developmentally regulated, representing ~ 27% of the stage-regulated transcriptome. Their expression was generally higher in amastigotes than in promastigotes, highlighting their importance in parasite intracellular development. Protein prediction tools combined to mass-spectrometry revealed that 7.6% of these lncRNAs have a limited protein-coding potential.

Conclusions: This is the first comprehensive transcriptomic analysis of developmental stages using single-molecule Nanopore DRS. Our findings advance knowledge on existing expression datasets and provide new insights into the transcriptome complexity and dynamics of both protein-coding and non-coding sequences throughout the parasite development.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12864-025-11767-8.