The human CD45 gene encodes a protein-tyrosine phosphatase that exhibits differential isoform expression in resting and activated T cells due to alternative splicing of three variable exons. Previously, we have used biochemical methods to identify two regulatory proteins, hnRNP L and PSF, which contribute to the activation-induced skipping of CD45 via the ESS1 regulatory element in variable exon 4. Here we report the identification of a third CD45 regulatory factor, hnRNP L-like (hnRNP LL), via a cell-based screen for clonal variants that exhibit an activation-like phenotype of CD45 splicing even under resting conditions. Microarray analysis of two splicing-altered clones revealed increased expression of hnRNP LL relative to wild-type cells. We further demonstrate that both the expression of hnRNP LL protein and its binding to ESS1 are up-regulated in wild-type cells upon activation. Forced overexpression of hnRNP LL in wild-type cells results in an increase in exon repression, while knockdown of hnRNP LL eliminates activation-induced exon skipping. Interestingly, analysis of the binding of hnRNP L and hnRNP LL to mutants of ESS1 reveals that these proteins have overlapping, but distinct binding requirements. Together, these data establish that hnRNP LL plays a critical and unique role in the signal-induced regulation of CD45 and demonstrate the utility of cell-based screens for the identification of novel splicing regulatory factors.

Cells can regulate their protein repertoire in response to extracellular stimuli via alternative splicing; however, the mechanisms controlling this process are poorly understood. The CD45 gene undergoes alternative splicing in response to T-cell activation to regulate T-cell function. The ESS1 splicing silencer in CD45 exon 4 confers basal exon skipping in resting T cells through the activity of hnRNP L and confers activation-induced exon skipping in T cells via previously unknown mechanisms. Here we have developed an in vitro splicing assay that recapitulates the signal-induced alternative splicing of CD45 and demonstrate that cellular stimulation leads to two changes to the ESS1-bound splicing regulatory complex. Activation-induced posttranslational modification of hnRNP L correlates with a modest increase in the protein's repressive activity. More importantly, the splicing factor PSF is recruited to the ESS1 complex in an activation-dependent manner and accounts for the majority of the signal-regulated ESS1 activity. The associations of hnRNP L and PSF with the ESS1 complex are largely independent of each other, but together these proteins account for the total signal-regulated change in CD45 splicing observed in vitro and in vivo. Such a combinatorial effect on splicing allows for precise regulation of signal-induced alternative splicing.To maintain viability most, if not all, cells within an organism must be capable of responding to a changing environment. For example, neuronal and muscle cells must respond to activation to promote behaviors and movement, while lymphoid cells must respond to an immune challenge to prevent or control infection. Such flexibility requires that individual cells have the ability to change function rapidly and precisely in response to a given stimulus. In general, cellular responsiveness is accomplished through the activity of signal transduction cascades that ultimately alter protein expression or function via numerous mechanisms. Much of the previous work on signal-regulated protein expression has focused on the regulation of transcription; however, there is growing recognition of the abundance and importance of signal-induced changes in alternative splicing (2, 11, 13).Alternative splicing is the process by which exons of a given gene are differentially included or excluded from the final message, such that a single gene can be processed into multiple discrete mRNAs (3, 16). Since each of these variant mRNAs typically encodes a unique protein of distinct function, changes in the splicing pattern of a gene can have a profound effect on protein expression and cellular activity. It has recently become apparent that numerous genes undergo changes in splicing patterns upon neuronal depolarization (2, 13) or T-cell activation (11), suggesting a broad role for this mode of gene regulation in determining the physiologic response of cells to stimulation. Thus, understanding the mechanisms by which extracellular stimuli can induce changes in the splicing regulatory machinery is of prima...

The HMG-box transcription factor LEF1 controls many developmentally regulated genes, including genes that activate expression of the T-cell antigen receptor alpha chain (TCR-alpha) in developing thymocytes. At least two distinct isoforms of LEF1 are expressed, resulting from variable inclusion of LEF1 exon 6; however, the expression pattern of these isoforms and mechanism of splicing regulation have not been explored. Here we demonstrate that inclusion of LEF1 exon 6 is increased during thymic development and in response to signaling in a cultured T-cell line in a manner which temporally correlates with increased expression of TCR-alpha. We further find that inclusion of exon 6 is dependent on the signal-induced increase in expression and binding of the splicing factor CELF2 to two intronic sequences flanking the regulated exon. Importantly, loss of exon 6 inclusion, through knockdown of CELF2 or direct block of the exon 6 splice site, results in reduced expression of TCR-alpha mRNA. Together, these data establish the mechanistic basis of LEF1 splicing regulation and demonstrate that LEF1 alternative splicing is a contributing determinant in the optimal expression of the TCR-alpha chain.

Alternative splicing is typically controlled by complexes of regulatory proteins that bind to sequences within or flanking variable exons. The identification of regulatory sequence motifs and the characterization of sequence motifs bound by splicing regulatory proteins have been essential to predicting splicing regulation. The activation-responsive sequence (ARS) motif has previously been identified in several exons that undergo changes in splicing upon T cell activation. hnRNP L binds to this ARS motif and regulates ARS-containing exons; however, hnRNP L does not function alone. Interestingly, the proteins that bind together with hnRNP L differ for different exons that contain the ARS core motif. Here we undertake a systematic mutational analysis of the best characterized context of the ARS motif, namely the ESS1 sequence from CD45 exon 4, to understand the determinants of binding specificity among the components of the ESS1 regulatory complex and the relationship between protein binding and function. We demonstrate that different mutations within the ARS motif affect specific aspects of regulatory function and disrupt the binding of distinct proteins. Most notably, we demonstrate that the C77G polymorphism, which correlates with autoimmune disease susceptibility in humans, disrupts exon silencing by preventing the redundant activity of hnRNPs K and E2 to compensate for the weakened function of hnRNP L. Therefore, these studies provide an important example of the functional relevance of combinatorial function in splicing regulation and suggest that additional polymorphisms may similarly disrupt function of the ESS1 silencer.Proper control of protein expression is essential for human health and development. A critical step in determining protein expression is that of pre-mRNA splicing in which non-coding intronic sequences are removed and exonic sequences are joined together through the action of the spliceosome (1). Importantly, the pattern of pre-mRNA splicing for any given gene is not a static choice; rather the inclusion or exclusion of an exon or group of exons can be highly variable (2, 3). Such alternative splicing of an exon is dictated by splicing enhancer or silencer sequences located within the exon or the flanking introns. These regulatory sequences typically bind to trans-acting protein factors that, in turn, interact with components of the spliceosome in such a way that promotes or inhibits the activity of the spliceosome on the exon substrate (2, 3).Alternative splicing alters the protein coding potential of the vast majority of human genes, often in a cell type-specific manner or in response to environmental cues (4, 5). Alternative splicing is especially prevalent in genes expressed in the nervous and immune systems, in which functional diversity and cellular responsiveness are particularly critical (6). Disruption of normal alternative splicing in such cell types has been linked to an increasing number of human diseases, underscoring the physiologic significance of this mode of gene regulation ...

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