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.