Initiation codon context governs translation-coupled mRNA decay and coordinated expression in the human parasite Leishmania

In the absence of canonical, promoter-based transcriptional regulation, Leishmania has evolved alternative regulatory mechanisms for adaptive gene expression, including post-transcriptional control via differential mRNA turnover. While this mechanism is recognized as critical in Leishmania , fundamental aspects of transcript stability in these parasites remain to be elucidated, such as the role of translation initiation-mediated mRNA decay. We addressed this important gap by investigating the role of the initiation codon context (Kozak sequence) in gene expression in L. donovani . Mapping Kozak sequences across the trypanosomatid genomes revealed important differences in nucleotide preference across the genus and sub-genus levels, suggesting important cis -regulatory function. Within a single species, only a small subset of possible Kozak sequences is associated with several start codons, further supporting their role in expression control. Transgenic L. donovani lines expressing EGFP under the control of distinct Kozak variants indeed demonstrated that the nucleotide context of the start codon directly modulates both protein expression and mRNA stability, which was associated with increased recruitment of mRNA to heavy polysomes. Parasite exposure to the translation inhibitor cycloheximide restored EGFP expression driven by a weak Kozak sequence, revealing a direct link between mRNA stability and Kozak-mediated translatability. RNA-seq analysis of parasites arrested for transcription or translation elongation revealed transcripts enriched for the GO terms ‘RNA modification’ and ‘pseudouridine synthesis’ as key targets for translation-dependent mRNA turnover. The segregation of these transcripts into functional clusters with distinct Kozak profiles further suggests that Kozak sequence composition defines Kozak-governed regulons in Leishmania. Within this regulatory framework, the -3 nucleotide is identified as the key positional determinant driving differential transcript abundance. Our work uncovers a key role for translation initiation-coupled mRNA decay in Leishmania gene expression regulation adding a previously underappreciated layer of post-transcriptional regulation in parasite adaptation.