Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway

Differential gene expression through alternative pre-mRNA splicing is crucial to various physiological and pathological conditions. Upon activation of B and T lymphocytes during an immune response, variant isoforms of the cell surface molecule CD44 are generated by alternative pre-mRNA splicing. We show here that in primary mouse T cells as well as in the murine LB-17 T-cell line upregulation of variant CD44 mRNA species upon T-cell activation requires activation of the MEK-ERK pathway. By employing mutant signaling molecules and a novel luciferase-based splice reporter system we demonstrate that the Ras-Raf-MEK-ERK signaling cascade, but not the p38 MAP-kinase pathway, activates a mechanism that retains variant CD44 exon v5 sequence in mature mRNA. The findings demonstrate that a highly conserved pleiotropic signaling pathway links extracellular cues to splice regulation, providing an avenue for tissue-specific, developmental or pathology-associated splicing decisions.     

Alternative splicing of CD45 pre-mRNA is uniquely obedient to conditions in lymphoid cells.

The leucocyte common antigen (LCA or CD45) consists of various isoforms generated by alternative splicing of variable exons 4, 5 and 6 (or A, B and C). To follow splicing behaviour in different cell types we developed a human CD45 mini-gene and analysed its expression in transfected cell lines and transgenic mouse tissues. In Cos-1, HeLa and 3T3 cells we found distinct expression patterns which could only be modulated slightly by protein synthesis inhibitors but not by variation in culture conditions like pH, serum concentration and cell density, or by stimulation with phorbol ester (TPA). In all non-lymphoid transgenic tissues the default splicing pattern (CD45R0) was found, while the expression profile in lymphoid cells, where all eight isoforms are present, mimics that of the endogenous mouse LCA gene products. Next, to examine the factors involved in alternative exon use we analysed the expression pattern of members of the family of SR proteins, well known splicing regulators with arginine/serine-rich (R/S) domains. Cell lines expressed variable levels of SRp75, SRp30 and SRp20 and constant amounts of SRp40. Mouse tissues expressed large amounts of SRp75, SRp55 and SRp40, additional expression of SRp30s and SRp20 was restricted to lymphoid tissues. Therefore, SRp30 and SRp20 may contribute to forming the appropriate cellular conditions for alternative use of CD45 exons 4-6 in the haematopoietic compartment.     

Trans-acting factors regulate the expression of CD44 splice variants.

Variant isoforms of the cell surface glycoprotein CD44 (CD44v) are expressed during development, in selected adult tissues and in certain metastatic tumor cells. CD44v differ from the standard isoform (CD44s) by up to ten additional exon sequences included by alternative splicing. By cell fusion experiments, we have obtained evidence for the existence of cell-type specific trans-acting factors recruiting CD44 variant exon sequences. Stable cell hybrids of CD44s and CD44v expressing cells indicated a dominant mechanism for variant-exon inclusion. In transient interspecies heterokaryons of human keratinocytes and rat fibroblasts, the ability of the keratinocytes to include all variant exon sequences in CD44 was conferred completely on the rat fibroblast nucleus. Fusions of cells with complex CD44 splice patterns do not permit interpretation of splice control by the relative abundance of a single trans-acting factor, but rather by (a) positively acting factor(s) recruiting variant exon sequences in the 3' to 5' direction and additional factors selecting individual exons. Since the pancreatic carcinoma cell line BSp73ASML (in contrast to the cervix carcinoma cell lines SiHa and ME180) could not transfer its specific splice pattern in cell fusions, we conclude that in some tumors, splicing is also controlled by mutation of cis-acting recognition sites.     

A model system for activation-induced alternative splicing of CD45 pre-mRNA in T cells implicates protein kinase C and Ras

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 activation-induced 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.     

Clathrin light chain B: gene structure and neuron-specific splicing.

The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing.     

Differential expression of the human CD8beta splice variants and regulation of the M-2 isoform by ubiquitination

The CD8alphabeta heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8(+) T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8(+) T cells. In total CD8(+) T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8(+) T cells. Upon stimulation of CD8(+) T cells, the M-2 variant mRNA levels were elevated 10-20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8beta fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8(+) T cell responses.     

Prolonged expression of high molecular mass CD45RA isoform during the differentiation of human progenitor thymocytes to CD3+ cells in vitro.

CD45, the leukocyte common Ag, has been shown to characterize T cell development both within the thymus and among peripheral T cells. The work reported here demonstrates that human multinegative (MN) thymocytes, depleted of cells bearing CD3, CD4, CD8, and CD19, express predominantly the high molecular mass CD45RA isoform, and lack low molecular mass CD45RB isoforms and CD45R0 as detected by immunofluorescence. By immunoprecipitation of surface-labeled CD45 molecules from MN thymocytes, a proportion of the CD45 is in fact of low molecular mass but does not include epitopes recognized by CD45R0, nor by CD45RB mAb specific for the p190. This suggests either glycosylation variants of CD45RB/CD45R0 undetectable by our mAb, or underglycosylated CD45RA. MN thymocytes lack TCR-alpha beta mRNA confirming their early developmental stage. Upon culture with IL-2 or with mitogenic combinations of anti-CD2/CD28 mAb, MN thymocytes differentiate to acquire CD3, TCR-alpha beta, and in some cases CD4 and/or CD8. We have predicted that maintenance of CD45RA and lack of CD45R0 expression is fundamental to generative thymic development. If correct, this demands that unlike peripheral T cells, differentiation of MN thymocytes should be accompanied by prolonged expression of high molecular mass CD45 isoforms. Analysis of CD45 isoform expression during MN thymocyte development confirms this prediction and indicates that expression of CD45RA is maintained, at increasing density, for at least 8 to 12 days of culture. Unlike peripheral blood T cells, this is accompanied by the gradual acquisition of firstly the p190 isoforms of CD45RB and later by CD45R0, resulting in a population of CD3+TCR-alpha beta cells coexpressing CD45RA/RBp190/R0. Dot blot analysis of mRNA from differentiating MN thymocytes indicates prolonged expression of mRNA encoding CD45 exons a, b, and c, again in contrast to peripheral T cells which lose all mRNA for alternatively spliced CD45 exons within the first 24 h poststimulation. This is discussed in the context of negative selection during thymic development and interconversion of T cell subsets.     

Multisite and bidirectional exonic splicing enhancer in CD44 alternative exon v3

The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3.     

Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons

Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence.     

Transitions from high to low molecular weight isoforms of CD45 (T200) involve rapid activation of alternate mRNA splicing and slow turnover of surface CD45R

The expression of CD45 isoforms and of CDw29 has been analyzed as a function of time by correlating cell surface phenotype with mRNA synthesis. After activation, T cells lose CD45R and acquire a high density of CD45 p180 and of CDw29. Throughout this transition the density of CD45 common determinants steadily increases, resulting in a net gain of surface CD45. The gradual loss of CD45R could reflect a rapid switch in CD45 mRNA splicing patterns followed by a slow loss of surface CD45R. Alternatively it could reflect a delayed activation of splicing to produce the 4.8-kb CD45 mRNA, or long lived 5.4-kb CD45 mRNA. Analysis of CD45 mRNA indicated that at 24 h postactivation, 5.4-kb CD45 mRNA is lost and only 4.8-kb mRNA is detectable. The amount of 4.8-kb mRNA increases until day 3 and then decreases somewhat. Thus our results support the interpretation that transitions in CD45 isoform mRNA expression occur within the first 24 h after activation and that the persistence of CD45R+ T cells until day 3 to 4 of culture results from slow turnover of surface CD45R glycoprotein.     

Phosphorylation-dependent regulation of PSF by GSK3 controls CD45 alternative splicing

Signal-induced alternative splicing of the CD45 gene in human T?cells is essential for proper immune function. Skipping of the CD45 variable exons is controlled, in large part, by the recruitment of PSF to the pre-mRNA substrate upon T?cell activation; however, the signaling cascade leading to exon exclusion has remained elusive. Here we demonstrate that in resting T?cells PSF is directly phosphorylated by GSK3, thus promoting interaction of PSF with TRAP150, which prevents PSF from binding CD45 pre-mRNA. Upon T?cell activation, reduced GSK3 activity leads to reduced PSF phosphorylation, releasing PSF from TRAP150 and allowing it to bind CD45 splicing regulatory elements and repress exon inclusion. Our data place two players, GSK3 and TRAP150, in the complex network that regulates CD45 alternative splicing and demonstrate a paradigm for signal transduction from the cell surface to the RNA processing machinery through the multifunctional protein PSF.     

Identification of cells deficient in signaling-induced alternative splicing by use of somatic cell genetics

In recent years, a growing number of mammalian genes have been shown to undergo alternative splicing in response to extracellular stimuli. However, the factors and pathways involved in such signal-induced alternative splicing are almost entirely unknown. Here we describe a novel method for identifying candidate trans-acting factors that are involved in regulating mammalian alternative splicing, using the activation-induced alternative splicing of the human CD45 gene in T cells as a model system. We generated a cell line that stably expresses a CD45 minigene-based GFP reporter construct, such that the levels of green-fluorescent protein (GFP) expressed in the cell reflect the splicing state of the endogenous CD45 gene. Following mutagenesis of this cell line, and multiple rounds of selection for cells that displayed aberrant levels of GFP expression, we isolated several cell lines that are at least partially defective in their ability to support regulated alternative splicing of endogenous CD45 pre-mRNA in response to cell stimulation. Thus we have successfully isolated mutants in a mammalian alternative splicing pathway through use of a somatic cell-based genetic screen. This study clearly demonstrates the feasibility of using genetic screens to further our understanding of the regulation of mammalian splicing, particularly as it occurs in response to environmental cues.