Abstract
Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as “central” interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, “peripheral” interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
Highlights
T cell activation is a central mechanism of the mammalian adaptive immune response to pathogenic stimuli
Isolated primary CD4 T cells from normal human donors are a model for T cell activation, but the availability of primary human cells limits their use in large-scale genomic studies
U2AF1 knockdown in activated CD4 T cells results in a dramatic decrease in binding for all tested proteins relative to the negative control siRNA (Fig 7A). These results demonstrate that U2AF1 is critical to the integrity of U2AF2 centered protein-protein complexes assembled with activation and not mediating binding to RNA via 3’ splice sites
Summary
T cell activation is a central mechanism of the mammalian adaptive immune response to pathogenic stimuli. The gene expression changes that occur as a result of transcription factor activation and binding are recognized as critical to the downstream immune response and lineage specification of the T helper effector subsets such as Th1, Th2, Th17, and Treg [4]. In a survey of 60 immune-related genes that undergo alternative splicing during activation of CD4 T cells, 7 different RNA binding proteins were linked to specific alternative splicing events [9]. A recent paper identified the role of the splicing factor, CELF2, in regulating alternative splicing after PMA activation of the JSL1 leukemic CD4 T cell line and employed several similar approaches to our present work including sequencing after CELF2 knockdown to identify splicing changes [11]. We will explore the value contributed by topdown studies in the context of the role in CD4 T cell activation-induced gene expression that includes changes in both canonical and alternative splicing
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