Oral Presentation 45th Lorne Genome Conference 2024

Site-specific RNA modification editing reveals m6A RNA methylation controls expression of the NLRP3 inflammasome sensor. (#32)

Natalia Pinello 1 , Quintin Lee 2 , Renhua Song 1 , Xi Yang 3 , Majid Mehravar 1 , Jessica Tieng 1 , Chinh Ngo 4 , Xing Huang 5 , Zhou-Li Cheng 5 , Mark Larance 1 , Fei Lan 5 , Ben Roediger 2 , Jocelyn Widagdo 6 , Victor Anggono 6 , Oliver Rausch 7 , Davinder Singh-Grewal 8 , Jeffrey Chaitow 8 , Xiangjian Zheng 3 , Dan Ye 5 , Si Ming Man 4 , Justin Wong 1
  1. School of Medical Sciences, University of Sydney, Camperdown, NSW, Australia
  2. Centenary Institute, Camperdown, NSW, Australia
  3. School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
  4. The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
  5. Fudan University, Shanghai, China
  6. Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia
  7. Storm Therapeutics Ltd, Cambridge, United Kingdom
  8. Department of Rheumatology, The Sydney Children Hospital Network, Sydney, NSW, Australia

N6-methyladenosine (m6A), the most abundant modification to mRNAs installed by the m6A methyltransferase, METTL3, mediates essential molecular processes including splicing, mRNA decay and translation. While m6A regulates fundamental cellular processes, its roles in the immune response are largely unknown. Central to innate immune response, signalling complexes called inflammasomes are activated in response to infection, cellular stress and other environmental cues. The NLRP3 inflammasome is a well-known cytosolic sensor that mediates caspase-1-dependent maturation and release of the pro-inflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation has been identified as a driver of many inflammatory disorders, including autoinflammatory, cardiovascular and neurodegenerative diseases. Therefore, enormous efforts have been invested in understanding the molecular mechanisms driving the NLRP3 pathway. To date, the specific role of METTL3 and RNA m6A methylation during inflammasome activation remains unclear. Using genetic manipulation and pharmacological inhibition, we demonstrate that METTL3 is essential to activate NLRP3 inflammasome in vitro and in vivo. We show that pharmacological inhibition of METTL3 attenuates NLRP3 hyperactivation in peripheral blood mononuclear cells isolated from Cryopyrin Associated Periodic Syndrome (CAPS) patients. Furthermore, METTL3 ablation in the myeloid compartment rendered mice relatively more resistant to NLRP3-mediated endotoxic shock. Combining transcriptome-wide m6A mapping and programmable CRISPR-based genomic and m6A editing techniques, we identified NLRP3 amongst the direct targets of METTL3 in macrophages as well as specific m6A sites that control the expression of NLRP3. Mechanistically, we found that site-specific METTL3-mediated m6A methylation regulates NLRP3 gene expression by i) co-transcriptionally promoting H3K9me2 demethylation near the NLRP3 promoter; and ii) promoting NLRP3 translation via the m6A reader protein, YTHDF1. Overall, our study places m6A-mediated regulation of gene expression at the centre of immunity and brings forward METTL3 and YTHDF1 as potential targets for therapeutic intervention in inflammation-driven syndromes.