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
Bibliographic record
Abstract
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 trans-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 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 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 L. infantum transcriptome expresses a rich repertoire of 1,825 lncRNAs, of which 98% were not previously annotated in L. infantum and only 21.5% were found in L. major. These lncRNAs exhibit generally distinct expression patterns from the 3’UTRs they derived and several are developmentally regulated, representing ~ 27% of the L. infantum 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 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 both protein-coding and non-coding sequences throughout the parasite development.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".