Aberrant splicing in Huntington’s disease accompanies disrupted TDP-43 activity and altered m6A RNA modifications [eCLIP-seq]

Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here, we identify the RNA-binding TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Dysregulated nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 is shown to be present in HD mice and human brains with TDP-43 co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions and from previously observed TDP-43 pathologies. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum from HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD and highlights the critical nature of TDP-43 loss of function across multiple neurodegenerative diseases. To investigate the epitranscriptomic landscape in the HD R62 mouse model, we carried out m6A-eCLIP sequencing using Eclipse Bioinnovations's m6A-eCLIP sequencing kit. We used both males and female mice n=4 each per genotype, and dissected out cortex and striatum for downstream library construction. To investigate TDP-43's RNA targets in the HD R62 mouse model, we carried out m6A-eCLIP sequencing using Eclipse Bioinnovations's RBP-eCLIP sequencing kit. We used both males and female mice n=4 each per genotype, and dissected out cortex and striatum for downstream library construction. To investigate the effect of TDP-43 knockdown on the m6A landscape we knocked down TDP-43 via ASO followed by m6A-eCLIP seq. n=3 per genotype was used. Please note that processed data files generated from both input and IP samples are linked to the corresponding IP sample records.