m⁶A landscape is more pervasive when Trypanosoma brucei exits the cell cycle

N6-methyladenosine (m⁶A) is an 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 the m⁶A landscape varies across the life cycle remains poorly defined. Using full-length, non-fragmented RNA, we immunoprecipitated and sequenced m⁶A-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 m⁶A 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 m⁶A, 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 m⁶A regulatory landscape is specific to each life cycle stage, becoming more pervasive as T. brucei exits the cell cycle.