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

Abstract 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. N 6 -methyladenosine (m 6 A) is an abundant post-transcriptional modification that promotes the decay of messenger RNAs in the cytosol. However, how recognition of m 6 A sites is integrated with other regulatory mechanisms that alter the fate of immunoregulatory mRNAs in CD8 + T cells remains unexplored. Here, we apply the m 6 A-iCLIP and GLORI methods to identify the importance of m 6 A sites flanked by AU-rich elements (AREs) within the 3’UTRs of CD8 + T cell mRNAs. Presence of such ARE-flanking m 6 A 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 m 6 A 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 m 6 A and ARE-dependent mechanisms in CD8 + T cells, providing new approaches for modulating mRNA decay in T cell activation.