Meta-unstable mRNAs in activated CD8+ T cells are defined by interlinked AU-rich elements and m6A mRNA methylation.

CD8+ T cells can rapidly produce effector molecules following activation. This activation triggers rapid changes in gene expression that rely on the control of mRNA levels via multiple mechanisms, including RNA modifications. N6-methyladenosine (m6A) is an abundant post-transcriptional modification that promotes the decay of messenger RNAs in the cytosol. However, how recognition of m6A sites is integrated with other regulatory mechanisms that alter the fate of immunoregulatory mRNAs in CD8+ T cells remains unexplored. Here, we apply the m6A-iCLIP and GLORI methods to identify the importance of m6A sites flanked by AU-rich elements (AREs) within the 3'UTRs of CD8+ T cell mRNAs. Presence of such ARE-flanking m6A motifs predicts meta-unstable mRNAs that rapidly decay upon CD8+ T cell activation. We demonstrate interdependent effects of mutations in the identified AREs and RRACHs on TNF mRNA stability. The ARE-flanking m6A sites in these mRNAs show particularly high iCLIP crosslinking of YTHDF proteins, which are also identified by proteomic interactome analyses along with additional novel RNA-binding proteins. Our study reveals a crosstalk between m6A and ARE-dependent mechanisms in CD8+ T cells, providing new approaches for modulating mRNA decay in T cell activation.