A Model System for Activation-Induced Alternative Splicing of CD45 Pre-mRNA in T Cells Implicates Protein Kinase C and Ras

Abstract

Multiple isoforms of the protein tyrosine phosphatase CD45 are expressed on the surface of human T cells. Interestingly, the expression of these isoforms has been shown to vary significantly upon T-cell activation. In this report, we describe a novel cell line-based model system in which we can mimic the activation-induced alternative splicing of CD45 observed in primary T cells. Of the many proximal signaling events induced by T-cell stimulation, we show that activation of protein kinase C and activation of Ras are important for the switch toward the exclusion of CD45 variable exons, whereas events related to Ca 2؉ flux are not. In addition, the ability of cycloheximide to block the activation-induced alternative splicing of CD45 suggests a requirement for de novo protein synthesis. We further demonstrate that sequences which have previously been implicated in the tissue-specific regulation of CD45 variable exons are likewise necessary and sufficient for activationinduced splicing. These results provide an initial understanding of the requirements for CD45 alternative splicing upon T-cell activation, and they confirm the importance of this novel cell line in facilitating a more detailed analysis of the activation-induced regulation of CD45 than has been previously possible.Alternative pre-mRNA splicing is a process by which multiple functionally distinct proteins may be encoded from a single gene through the variable inclusion of individual exons. In general, variable exon inclusion is regulated by proteins which bind to sequences within the regulated gene and influence the recognition of splice sites by the basic splicing machinery, or spliceosome (for recent reviews, see references 1, 4, 6, and 18). Frequently, alternative splicing is regulated in a tissue-or developmental stage-specific fashion through the activity of tissue-specific factors (17, 60). In addition, there have been several documented examples of exon inclusion being regulated in response to extracellular stimuli, including stimulation of the T-cell receptor (TCR) (10,21,47,51,61,65). However, the mechanisms by which such signaling events can influence premRNA splicing are largely unexplored.Engagement of the TCR by ligand initiates a signal transduction cascade within the T cell which ultimately results in a number of morphological and functional changes in the cell (11,62). One such change is a dramatic alteration in the cell surface expression of isoforms of the transmembrane protein tyrosine phosphatase CD45 (for reviews see references 57 and 58). The gene encoding CD45 encompasses 33 exons. Most of these exons, including those which encode the intracellular phosphatase domains, are constitutively included in the mature mRNA. However, three of the exons which encode part of the extracellular domain (exons 4, 5, and 6) are variably excluded from the mRNA. The peptide sequences encoded by the CD45 variable exons are rich in O-linked glycosylation sites; thus, a change in inclusion of these exons from the mRNA results in a dramatic change in t...

Control of alternative pre-mRNA splicing by Ca++ signals

“…In some cases, it takes several hours for the splice variant mRNA to change significantly, and it has been shown that de novo protein synthesis is required [97,163,164]. Interestingly, the nuclear level of the hnRNP A1 protein is increased in depolarized neurons in regulating the exon 21 (or called C1 cassette exon) of NMDAR1 [72,84].…”

Activation-induced Tumor Necrosis Factor Receptor-associated Factor 3 (Traf3) Alternative Splicing Controls the Noncanonical Nuclear Factor κB Pathway and Chemokine Expression in Human T Cells

“…Over the past years, the Jurkat-derived Jsl1 T cell line has become a prime model system to investigate activation induced alternative splicing (30,31). A recent RNA-Seq approach in Jsl1 cells suggested an inducible switch in Traf3 isoform expression (3).…”

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

“…1B, lanes 2 and 5), however, TPA is known to regulate the splicing of other alternative exons, particularly those of the cell surface molecules CD44 and CD45 [5,6]. We thus tested TPA with more cell lines for Bcl-x splicing changes.…”

“…Many splice variants have distinct functions and can be regulated by extracellular factors, but the molecular bases of the regulations remain largely unclear [1,4]. In several cases, pre-mRNA elements involved in responses to external factors have been identified [5][6][7][8][9]. In one case, the extracellular signal-regulated kinase (ERK) was found to directly phosphorylate the splicing factor Sam68 in regulating CD44 splicing [10].…”