Abstract N6-methyladenosine (m 6 A) is a mRNA modification with important roles in gene expression. In African trypanosomes, this post-transcriptional modification is detected in hundreds of transcripts and it affects the stability of the variant surface glycoprotein (VSG) transcript in the proliferating blood stream form. However, how m6A landscape varies across the life cycle remains poorly defined. Using full-length, non-fragmented RNA, we immunoprecipitated and sequenced m 6 A-modified transcripts across three life cycle stages of Trypanosoma brucei – slender (proliferative), stumpy (quiescent), and procyclic forms (proliferative). We found that 1037 transcripts are methylated in at least one of these three life cycle stages. While 21% of methylated transcripts are common in the three stages of the life cycle, globally each stage has a distinct methylome. Interestingly, 47% of methylated transcripts are detected in the quiescent stumpy form only, suggesting a critical role for m 6 A when parasites exit the cell cycle and prepare for transmission by the Tsetse fly. In this stage, we found that a significant proportion of methylated transcripts encodes for proteins involved in RNA metabolism, which is consistent with their reduced transcription and translation. Moreover, we found that not all major surface proteins are regulated by m 6 A, as procyclins are not methylated, and that, within the VSG repertoire, not all VSG transcripts are demethylated upon parasite differentiation to procyclic form. This study reveals that the m 6 A regulatory landscape is specific to each life cycle stage, becoming more pervasive as T. brucei exits the cell cycle. Summary African trypanosome parasites adapt to mammalian and insect hosts by adjusting gene expression, morphology, and metabolism. In this study, we focus on how N6-methyladenosine (m 6 A), a post-transcriptional modification, affects the parasite’s transcriptome throughout its differentiation from the mammalian host to the fly. We found that methylation is differentially regulated as the life cycle progresses, being particularly prevalent in the non-proliferative stumpy form, as more methylated transcripts are found at this insect-infective stage than in slender and procyclic forms. We further show that the not all parasite surface proteins are regulated by m 6 A and that the previously identified link between m 6 A methylation and the expression level of the major surface protein of bloodstream forms applies to the active variant surface glycoprotein, but not always to silent genes, suggesting two distinct regulatory mechanisms of (de)methylation.

m6A landscape is more pervasive whenTrypanosoma bruceiexits the cell cycle