Cell type specific regulation of m6A modified RNAs in the aging brain

The aging brain is highly vulnerable to cellular stress, and neurons often employ numerous mechanisms to combat neurotoxic proteins and promote healthy brain aging. The RNA modification m6A has been shown to be a critical regulator of RNA stability and translation in cells during stress. m6A is highly enriched in the Drosophila brain and is critical for the acute heat stress response. Here we examine m6A response to chronic stresses of aging and degenerative disease. In the brain, m6A levels dynamically increased with age and disease, marking critical signaling pathway transcripts that become downregulated in age and disease. Unexpectedly, there is opposing regulation of m6A transcript translation in neural vs glial cells, which conferred different outcomes on animal healthspan with Mettl3 knockdown to reduce m6A. Moreover, these data reveal that knockdown of Mettl3 in glial tauopathy is beneficial, leading to increased animal survival. These findings provide mechanistic insight into regulation of m6A modified transcripts with age and disease that varies based on cell type. Overall design: head m6A-IP, total brain RNA-seq, FACS sorted Repo GFP+/- or Elav GFP+, and TRAP IP from Repo or Elav cells expressing UAS-RPL3-FLAG tag used to investigate the role of the m6A RNA modification in age and with AB expression and Mettl3 knockdown. For experiment 21 and 08, m6A-IPseq libraries were made using SMARTer Stranded Total RNA-Seq Kit V3 without rRNA depletion (takarabio, 634486) for IPed and input RNA. Experiment 07 brain RNA-seq libraries were prepared using the Tru-seq stranded mRNA library prep. Experiment 25, 27, and 29 FACS sorting and TRAP-seq libraries were made using SMART-Seq V4 + NexteraXT Library Preparation. All libraries were sequenced using illumina Illumina NovaSeq 6000 with 40M paired end reads (2x150bp).