Multiomics analysis couples mRNA turnover and translational control of glutamine metabolism to the differentiation of the activated CD4+ T cell

The ZFP36 family of RNA-binding proteins acts post-transcriptionally to repress translation and promote RNA decay. Studies of genes and pathways regulated by the ZFP36 family in CD4+ T cells have focussed largely on cytokines, but their impact on metabolic reprogramming and differentiation is unclear. Using CD4+ T cells lacking Zfp36 and Zfp36l1, we combined the quantification of mRNA transcription, stability, abundance and translation with crosslinking immunoprecipitation and metabolic profiling to determine how they regulate T cell metabolism and differentiation. Our results suggest that ZFP36 and ZFP36L1 act directly to limit the expression of genes driving anabolic processes by two distinct routes: by targeting transcription factors and by targeting transcripts encoding rate-limiting enzymes. These enzymes span numerous metabolic pathways including glycolysis, one-carbon metabolism and glutaminolysis. Direct binding and repression of transcripts encoding glutamine transporter SLC38A2 correlated with increased cellular glutamine content in ZFP36/ZFP36L1-deficient T cells. Increased conversion of glutamine to α-ketoglutarate in these cells was consistent with direct binding of ZFP36/ZFP36L1 to Gls (encoding glutaminase) and Glud1 (encoding glutamate dehydrogenase). We propose that ZFP36 and ZFP36L1 as well as glutamine and α-ketoglutarate are limiting factors for the acquisition of the cytotoxic CD4+ T cell fate. Our data implicate ZFP36 and ZFP36L1 in limiting glutamine anaplerosis and differentiation of activated CD4+ T cells, likely mediated by direct binding to transcripts of critical genes that drive these processes. mRNA-seq (3 biological replicates), nascent, 4sU-labelled mRNA-seq (2 biological replcates, separated into labelled [nascent RNA], unlabelled [pre-existing RNA] and total fractions) and Ribo-seq (5 biological replicates) were performed in activated CD4+ T cells from control and Zfp36/Zfp36l1 double conditional knockout mice. To identify direct targets of ZFP36-family proteins, iCLIP was performed on activated CD4+ T cells from WT C57BL/6 mice (3 biological replicates), using two independent antibodies that bind to ZFP36L1.