m6A RNA modification of mHttintron 1 regulates the generation ofHtt1ain Huntington’s Disease

Abstract

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expanded, somatically unstable CAG repeat in the first exon of the huntingtin gene (HTT). In the presence of an expanded CAG repeat, huntingtin mRNA undergoes an aberrant processing that generatesHTT1atranscripts with exon 1 and intron 1 sequences, which encodes the aggregation-prone and pathogenic HTTexon 1 protein. The regulatory mechanisms that contribute to the production ofHTT1aare not fully understood. In a previous transcriptome-wide m6A landscape study performed inHdh+/Q111knock-in mice, we have found that the proximal region of intron 1 to exon1-intron 1 splice site inHttRNA is highly modified by m6A. Several pieces of evidence have demonstrated that m6A is involved in RNA processing and splicing. Therefore, in this study we set out to explore the impact of m6A RNA modifications in the generation ofHtt1a. We show in the striatum ofHdh+/Q111mice that m6A is enriched in intronic sequences 5’ to the cryptic poly (A) sites (IpA1 and IpA2) at 680 and 1145 bp into intron 1 as well as inHtt1apolyadenylated mRNA. We also verified the presence of specific m6A-modified sites near the 5’ exon1-intron1 splice donor site. IntronicHTTm6A methylation was recapitulated in human samples showing a significantly increased methylation ratio in HD putamenpost-mortemsamples and in HD fibroblast cell lines from pre-symptomatic and symptomatic patients. In order to test the hypothesis that the m6A modification is involved in mutantHttRNA processing, we performed a pharmacological inhibition of METTL3 and a targeted demethylation ofHttintron 1 in HD cells using a dCas13-ALKBH5 system. We found thatHtt1atranscript levels in HD cells are regulated by METTL3 and by methylation status inHttintron 1. Site-specific manipulation with an RNA editing system resulted in decreased expression levels ofHtt1a, which was accompanied by a reduction in DNA damage, a major hallmark in HD. Finally, we propose that m6A methylation in intron 1 is likely dependent on the expanded CAG repeats. These findings provide insight into the role of m6A in the generation of the aberrantly spliced mutantHtttranscripts with important implications for therapeutic strategies.