Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development

Abstract

Accumulating evidence has revealed significant roles for N6-methyladenosine (m 6 A) modification in the development of various cancers. We previously demonstrated an oncogenic role of m 6 A-modified CUB domain containing protein 1 (CDCP1) in bladder cancer (BC) progression. However, the biological functions and underlying molecular mechanisms of engineered programmable m 6 A modification of CDCP1 mRNA in BC remain obscure. Here, we established a targeted m 6 A RNA methylation system by fusing the catalytic domain of methyltransferase like 3 (METTL3CD) to RCas9 as the RNA-targeting module. The constructed RCas9- METTL3 retained methylation activity and mediated efficient site-specific m 6 A installation in the presence of a cognate single guide RNA and short protospacer adjacent motif-containing ssDNA molecule. Subsequently, targeting m 6 A installation onto the 3′ untranslated region of CDCP1 promoted CDCP1 mRNA translation and facilitated BC development in vitro and in vivo. Our findings demonstrate that the RCas9-METTL3 system mediates efficient sitespecific m 6 A installation on CDCP1 mRNA and promotes BC development. Thus, the RCas9-METTL3 system provides a new tool for studying m 6 A function and a potential strategy for BC epitranscriptome-modulating therapies.