m6A modification of mutant huntingtin RNA promotes the biogenesis of pathogenic huntingtin transcripts.

In Huntington's disease (HD), aberrant processing of huntingtin (HTT) mRNA produces HTT1a transcripts that encode the pathogenic HTT exon 1 protein. The mechanisms behind HTT1a production are not fully understood. Considering the role of m⁶A in RNA processing and splicing, we investigated its involvement in HTT1a generation. Here, we show that m⁶A methylation is increased before the cryptic poly(A) sites (IpA1 and IpA2) within the huntingtin RNA in the striatum of Hdh+/Q111 mice and human HD samples. We further assessed m⁶A's role in mutant Htt mRNA processing by pharmacological inhibition and knockdown of METTL3, as well as targeted demethylation of Htt intron 1 using a dCas13-ALKBH5 system in HD mouse cells. Our data reveal that Htt1a transcript levels are regulated by both METTL3 and the methylation status of Htt intron 1. They also show that m⁶A methylation in intron 1 depends on expanded CAG repeats. Our findings highlight a potential role for m⁶A in aberrant splicing of Htt mRNA. Synopsis: This study unveils that m⁶A RNA modification contributes to aberrant Htt splicing in Huntington's disease models, promoting the generation of the pathogenic Htt1a variant. Increased m6A methylation levels in Htt1a are detected at early stages in the striatum of Hdh^+/Q111 mice. HD mice and human samples reveal m⁶A methylation in the same intronic DRACH motif located in intron 1 of huntingtin RNA. Pharmacological inhibition and siRNA knockdown of METTL3, as well as targeted demethylation, reduce Htt1a expression in mouse cells. Blocking CAG repeats in exon 1 of Huntingtin RNA with LNA-CTG ASOs reduces m⁶A RNA methylation in intron 1 in mouse cells. This study unveils that m⁶A RNA modification contributes to aberrant Htt splicing in Huntington's disease models, promoting the generation of the pathogenic Htt1a variant. [ABSTRACT FROM AUTHOR]