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: Leishmania relies on posttranscriptional control to regulate gene expression. Protein-coding genes are synthesised as polycistronic precursors that are processed into individual mRNAs by coupled trans -splicing adding the spliced leader (SL) RNA to the 5'-end of all transcripts and 3' cleavage-polyadenylation. Here, we employ Nanopore direct RNA sequencing (DRS) combined with Illumina RNA-Seq to comprehensively interrogate the transcriptomes of Leishmania infantum developmental stages at single-molecule resolution. Results: Analysis of DRS full-length reads of poly(A)+-enriched RNA from L. infantum 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 boundaries and the length of UTRs. We observed a higher diversity for the poly(A) sites per transcript than the SL sites. The frequency of the primary SL site was ~ 64.2%, whereas the incidence of the primary poly(A) site was 24%, with most transcripts having additional poly(A) sites nearby. Alternative polyadenylation was detected in 11–13% of transcripts, of which 28% were developmentally regulated. Similarly, 26.5% of transcripts with different primary SL sites between developmental stages were differentially expressed. 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. Further, DRS uncovered multiple processing events occurring mostly within long 3'UTRs, leading to the formation of long non-coding RNAs (lncRNAs). The L. infantum transcriptome expresses a rich repertoire of 1,825 lncRNAs, of which 98% were not previously annotated in L. infantum and most are absent from L. major and may be specific to L. infantum . Generally, these lncRNAs exhibit distinct expression patterns from the 3'UTRs they derived, and several are developmentally regulated, representing 27% of the L. infantum stage-regulated transcriptome. Protein prediction tools combined to mass-spectrometry revealed that 7.6% of the lncRNAs have a limited protein-coding potential. Conclusions: This is the first comprehensive transcriptomic analysis of L. infantum developmental stages using single-molecule Nanopore DRS. Our findings advance knowledge on existing Leishmania expression datasets and provide new insights into the transcriptome complexity and dynamics of protein-coding and non-coding sequences throughout the parasite development.