Abstract
Alternative splicing of nuclear pre-mRNA is essential for generating protein diversity and regulating gene expression. While many immunologically relevant genes undergo alternative splicing, the role of regulated splicing in T cell immune responses is largely unexplored, and the signaling pathways and splicing factors that regulate alternative splicing in T cells are poorly defined. Here, we show using a combination of Jurkat T cells, human primary T cells, and ex vivo naïve and effector virus-specific T cells isolated after influenza A virus infection that SC35 phosphorylation is induced in response to stimulatory signals. We show that SC35 colocalizes with RNA polymerase II in activated T cells and spatially overlaps with H3K27ac and H3K4me3, which mark transcriptionally active genes. Interestingly, SC35 remains coupled to the active histone marks in the absence of continuing stimulatory signals. We show for the first time that nuclear PKC-θ co-exists with SC35 in the context of the chromatin template and is a key regulator of SC35 in T cells, directly phosphorylating SC35 peptide residues at RNA recognition motif and RS domains. Collectively, our findings suggest that nuclear PKC-θ is a novel regulator of the key splicing factor SC35 in T cells.
Highlights
Alternative splicing of nuclear pre-mRNA transcripts is an essential regulator of eukaryotic gene expression
Using a combination of Jurkat T cells, human primary T cells, and ex vivo naïve and effector virus-specific T cells isolated after influenza A virus infection, we show that SC35 phosphorylation (SC35p) is induced in response to stimulatory signals
Given that the splicing factor SC35 regulates alternative splicing in T cells, we initially determined the subcellular distribution of SC35 by fluorescence microscopy in the human Jurkat T cell line; cells were either non-stimulated or stimulated with phorbolmyristate acetate and calcium ionophore (PMA/I), a known protein kinase C (PKC) pathway inducer
Summary
Alternative splicing of nuclear pre-mRNA transcripts is an essential regulator of eukaryotic gene expression. Alternative splicing results in numerous functionally distinct protein isoforms from a single gene [1]. Pre-mRNA splicing takes place within the spliceosome, a ribonucleoprotein complex enriched in pre-mRNA splicing machinery including small nuclear ribonucleoproteins (snRNPs), spliceosome subunits, non-snRNP splicing factors, and a plethora of unknown mRNA-regulating nuclear factors [2, 3]. Upon target transcript binding at specific splice sites, spliceosomes catalyze the removal of non-coding introns and exon ligation to produce protein-coding mRNA. A number of mechanisms regulate alternative splicing of pre-mRNA, including exon skipping, intron retention, and the selective use of 3′ and 5′ splice sites [4]. Alternative splicing is a key mechanism for generating protein diversity and regulating gene expression and, plays an important role in cell function and development
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