A04 Role of RNA M6A modification in post-transcriptional regulation of mutant huntingtin expression. (12th September 2022)

• Record Type: Journal Article
• Authors: Pupak, Anika; Rodriguez-Navarro, Irene; Singh, Ankita; Ørom, Ulf Vang; Alberch, Jordi; Ginés, Silvia; Brito, Verónica
• Is Part Of: Journal of neurology, neurosurgery and psychiatry. Volume 93(2022)Supplement 1
• Page Start: A2
• Page End: A2
• Publication Date: 2022-09-12
• Subjects: Incomplete splicing -- RNA modifications -- N6-methyladenosine -- CRISPR/Cas13 -- gene therapy; Neurology -- Periodicals; Nervous system -- Surgery -- Periodicals; Psychiatry -- Periodicals; 616.8
• DOI: 10.1136/jnnp-2022-ehdn.4
• Languages: English
• ISSNs: 0022-3050

Abstract

Abstract : Background: The 'exon 1 fragment' of mutant HTT has been postulated as a key driver of neurotoxicity in Huntington´s Disease. It can be generated by proteolytic processing as well as by incomplete splicing of expanded-CAG-repeat HTT RNA. Although regulatory mechanisms that influence the amount of HTTexon1 production by incomplete splicing have been described, the impact of RNA modifications on the post-transcriptional regulation of HTT is unknown. Aims: To unravel the mechanisms involving N6-methyladenosine (m6A) in the post-transcriptional regulation of HTT. Methods: We performed MeRIP 3' RACE analysis for cryptic poly A sites to measure splicing in the striatum of HdhQ111/Q7 Knock in (KI) mice. We identified methylation motifs in both human and mouse models using a MazF-qPCR approach. Finally, we studied in vitro association between m6A modifications in the intronic region of mutant Htt and the generation of Htt exon 1 by targeting RNA demethylation in mutant Htt using a CRISPR/Cas13-based approach. Results: We have detected that the proximal region of intron 1 in Htt mRNA is heavily modified by m6A in HdhQ111/Q7 KI mice and human samples. Here we demonstrate in the striatum of KI mice m6A an enrichment in intronic sequences 5´ to the cryptic poly (A) sites but not in Full Length transcripts. Targeting RNA demethylation in mutant HTT, we showed that demethylation of the proximal region of Httexon1 leads to a significant reduction in Httexon1 transcripts levels. Conclusions: Our results support the involvement of m6A in the generation of aberrantly spliced mutant Htt transcripts.