N⁶-methyladenosine in poly(A) tails stabilize VSG transcripts

RNA modifications are important regulators of gene expression¹. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally². N⁶-methyladenosine (m⁶A) has been detected in this parasite, but its function remains unknown³. Here we found that m⁶A is enriched in 342 transcripts using RNA immunoprecipitation, with an enrichment in transcripts encoding variant surface glycoproteins (VSGs). Approximately 50% of the m⁶A is located in the poly(A) tail of the actively expressed VSG transcripts. m⁶A residues are removed from the VSG poly(A) tail before deadenylation and mRNA degradation. Computational analysis revealed an association between m⁶A in the poly(A) tail and a 16-mer motif in the 3' untranslated region of VSG genes. Using genetic tools, we show that the 16-mer motif acts as a cis-acting motif that is required for inclusion of m⁶A in the poly(A) tail. Removal of this motif from the 3' untranslated region of VSG genes results in poly(A) tails lacking m⁶A, rapid deadenylation and mRNA degradation. To our knowledge, this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a post-transcriptional mechanism of gene regulation.