Fast responders: uncovering immediate early splicing events following lymphocyte activation

Abstract

Lymphocytes require a certain time frame (24-48 hours) after their activation to fully develop effector functions. Hence, most studies have focused on characterizing splicing switches after this period. However, shortly after lymphocyte activation (0h to 4 hours), rapid gene expression changes occur, resulting in cytokines production and their secretion, as well as the initiation of cell proliferation, and differentiation. The expression of immediate early genes (IEG) is transiently induced upon T cell activation, with regulation being independent of de novo protein synthesis. This process relies on phosphorylation cascades that target the transcription machinery. In this project, we hypothesized that similar signaling cascades might also target the splicing machinery to induce immediate early splicing (IES). We aimed to delineate this concept and characterize its function in T cell and B cell activation. Furthermore, we investigated whether IES switches influence the translational machinery or whether they operate independently of this process. Through in silico bioinformatic analysis and biochemical wet lab experiments, we identified the rapid and transient retention of short introns among several genes within the first 1.5 hours of lymphocyte activation. This concerted splicing switch is independent of de novo protein synthesis. Further analysis showed its specific regulation by RAF/MEK/ERK1/2-mediated phosphorylation of the splicing factor - hnRNPC2. This hnRNPC isoform originates from an alternative 5’ splice site (A5SS) usage and contains 13 additional amino acids compared to hnRNPC1. Moreover, the T cell specificity of IES is preserved by the involvement of the kinase PKCθ, which is highly expressed in T cells. This is not the case in B cells, suggesting the involvement of another kinase in the hnRNPC2-controlled IES switch. IES affects many components of the translation apparatus, including transcripts encoding ribosomal proteins and translation factors, such as eukaryotic initiation factor eIF5A. Furthermore, we observed an hnRNPC2-dependent reduction in de novo translation during the hours immediately following T cell activation. We suggested that this reduction is mediated by the IES switch, as inducing the IES protein variant in eIF5A alone is sufficient to reduce translation globally. Altogether, we present a new paradigm for fast and transient alternative splicing regulation and suggest a fundamental role of IES in coordinating different layers of gene expression upon T cell activation.