GSE96052
ZFP36 RNA binding proteins restrain T-cell activation kinetics and anti-viral immunity [ribo-seq]
Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in T-cells, which confirmed regulation of cytokine expression and revealed unanticipated actions in regulating T-cell activation and proliferation. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, most robustly through a novel class of AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics by attenuating activation marker expression, limiting T-cell expansion, and promoting apoptosis in a cell autonomous manner. Strikingly, loss of ZFP36 in vivo accelerated T-cell responses to acute viral infection, and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T-cell expansion and effector functions, and suggest ZFP36 inhibition as a novel strategy to enhance immune-based therapies. Overall design: Ribosome profiling by was done for WT and Zfp36 KO CD4+ T-cells activated under Th1 polarizing conditions for 4 hours, to determine differentially translated mRNAs. Naïve CD4+ T-cells were purified with the CD4+CD62L+ isolation kit (Miltenyi) and incubated in co-cultures with formalin-fixed bone-marrow derived dendritic cells (BMDCs), anti-CD3e, 10 U/ml IL-2 and 5 ng/ml IL-12. After 4 hours, cells were harvested in the presence of cycloheximide and snap frozen in liquid nitrogen. Cytoplasmic lysates were prepared and treated with micrococcal nuclease, then monosomes were isolated by sucrose gradient fractionation as described in the accompanying protocol  Ribosome-protected-fragments (RPFs) were released from monosomes, and ligated to a pre-adenylated 3? linker (GTGTCAGTCACTTCCAGCGG), then amplified using the Br-dU incorporation, cDNA circularization, and PCR strategy described in detail in the accompanying publication.  RT primers were designed to incorporate sample indices for multiplexing (trimmed from uploaded sequences) and a 7 nucleotide degenerate sequence (NNNNNNN; present in uploaded reads) at the 5? end of the sequenced read. Amplified libraries were sequenced on the Illumina Miseq, with 75 nucleotide single-end reads. For analysis, adapter sequences were trimmed on both ends, samples were de-multiplexed using indices, and degenerate barcodes were appended to read IDs.  RPF reads were then aligned against the mouse Ensembl transcripts using Bowtie2.