Harald König

Signal-dependent regulation of splicing via phosphorylation of Sam68

Evolution of human organismal complexity from a relatively small number of genes—only approximately twice that of worm or fly—is explained mainly by mechanisms generating multiple proteins from a single gene, the most prevalent of which is alternative pre-messenger-RNA splicing. Appropriate spatial and temporal generation of splice variants demands that alternative splicing be subject to extensive regulation, similar to transcriptional control. Activation by extracellular cues of several cellular signalling pathways can indeed regulate alternative splicing. Here we address the link between signal transduction and splice regulation. We show that the nuclear RNA-binding protein Sam68 is a new extracellular signal-regulated kinase (ERK) target. It binds exonic splice-regulatory elements of an alternatively spliced exon that is physiologically regulated by the Ras signalling pathway, namely exon v5 of CD44. Forced expression of Sam68 enhanced ERK-mediated inclusion of the v5-exon sequence in mRNA. This enhancement was impaired by mutation of ERK-phosphorylation sites in Sam68, whereas ERK phosphorylation of Sam68 stimulated splicing of the v5 exon in vitro. Finally, Ras-pathway-induced alternative splicing of the endogenous CD44-v5 exon was abolished by suppression of Sam68 expression. Our data define Sam68 as a prototype regulator of alternative splicing whose function depends on protein modification in response to extracellular cues.

Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1 activity without altering AP-1 site occupation in vivo.

Phorbol esters stimulate and glucocorticoid hormones down‐regulate a variety of promoters such as that of the collagenase gene through the transcription factor AP‐1 (Fos/Jun). We now show by genomic footprinting of the collagenase promoter that phorbol ester treatment of cells results in the binding of AP‐1 to its cognate DNA binding site in vivo. The DNA‐protein contacts obtained in living cells are also found in vitro using cloned DNA and purified AP‐1. Although in vitro synthesized glucocorticoid receptor can disturb the DNA binding of Jun homodimers, it does not interfere with the binding of Fos‐Jun heterodimers or of purified AP‐1 in vitro. Consistently, fully inhibitory doses of glucocorticoid hormone cause no change in apparent occupation of the AP‐1 binding site in vivo. The hormone receptor acts without itself binding to DNA.

Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator.

Alternative splicing of pre‐mRNA is a fundamental mechanism of differential gene expression in that it can give rise to functionally distinct proteins from a single gene, according to the developmental or physiological state of cells in multicellular organisms. In the pre‐mRNA of the cell surface molecule CD44, the inclusion of up to 10 variant exons (v1–v10) is regulated during development, upon activation of lymphocytes and dendritic cells, and during tumour progression. Using minigene constructs containing CD44 exon v5, we have discovered exonic RNA elements that couple signal transduction to alternative splicing. They form a composite splice regulator encompassing an exon recognition element and splice silencer elements. Both type of elements are necessary to govern cell type‐specific inclusion of the exon as well as inducible inclusion in T cells after stimulation by concanavalin A, by Ras signalling or after activation of protein kinase C by phorbol ester. Inducible splicing does not depend on de novo protein synthesis. The coupling of signal transduction to alternative splicing by such elements probably represents the mechanism whereby splice patterns of genes are established during development and can be changed under physiological and pathological conditions.

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.

CD44 Acts Both as a Growth‐ and Invasiveness‐Promoting Molecule and as a Tumor‐Suppressing Cofactor

Abstract: High‐molecular‐weight splice variants of the CD44 transmembrane protein family have been implicated in tumorigenesis and metastasis formation. By contrast, in certain tumors‐for example, Burkitts lymphoma, neuroblastomas, and prostate cancer‐loss of CD44 expression seems to accompany transformation. Here we describe two modes of action of CD44 proteins. They can bind growth factors and present them to their authentic high‐affinity receptors, and thus promote proliferation and invasiveness of cells. Under these conditions the CD44 proteins recruit ERM proteins‐for example, ezrin or moesin‐to their cytoplasmic tails, thereby producing links to the cytoskeleton. This mode of action could account for the tumor‐promoting action of CD44 proteins. The second mode of action of CD44 proteins comes into play when cells reach confluent growth conditions. Under specific conditions, binding of another ligand, the ECM component hyaluronate, leads to the activation and binding to the CD44 cytoplasmic tail of the tumor suppressor protein merlin. The activation of merlin confers growth arrest, so‐called contact inhibition. This function of CD44 proteins defines them as tumor suppressors. The type of action of CD44 on a given cell will depend on the isoform pattern of CD44 expressed, on the cellular equipment with ERM protein members, on the nature of the ECM, and on yet‐unknown conditions.

Autoregulation of fos: the dyad symmetry element as the major target of repression.

Fos and Jun co‐operatively repress the fos promoter. Removal of all putative Fos/Jun binding sites from the fos promoter neither obliterates the repression by Fos/Jun in transient cotransfection experiments in NIH3T3 cells nor the turn‐off kinetics of serum‐induced fos expression in stably transfected NIH3T3 cells. The dyad symmetry element (DSE) suffices to subject a promoter to this type of repression. However, one of the putative Fos/Jun binding sites (‐292 to ‐299 and thus located immediately adjacent to the DSE), determines the very low level of basal expression.

Heterogeneous Ribonucleoprotein A1 Is Part of an Exon-specific Splice-silencing Complex Controlled by Oncogenic Signaling Pathways

Regulation of alternative pre-mRNA splicing, recognized as increasingly important in causing human disease, was studied using the CD44 gene, whose splice variants have been implicated in tumor progression. We identified heterogeneous ribonucleoprotein (hnRNP) A1 as a protein interacting in vitro and in vivo with regulatory splice elements in CD44 variant exon v5. Transient overexpression of hnRNP A1 prevented v5 exon inclusion, dependent on the exonic elements. HnRNP A1-dependent repression was exon-specific and could be relieved by coexpression of oncogenic forms of Ras and Cdc42. The results define hnRNP A1 as a decisive part of an oncogene-regulated splice-silencing complex, which can select between multiple alternatively spliced exons.

Splicing Segregation: The Minor Spliceosome Acts outside the Nucleus and Controls Cell Proliferation

The functional relevance and the evolution of two parallel mRNA splicing systems in eukaryotes--a major and minor spliceosome that differ in abundance and splicing rate--are poorly understood. We report here that partially spliced pre-mRNAs containing minor-class introns undergo nuclear export and that minor-class snRNAs are predominantly cytoplasmic in vertebrates. Cytoplasmic interference with the minor spliceosome further indicated its functional segregation from the nucleus. In keeping with this, minor splicing was only weakly affected during mitosis. By selectively interfering with snRNA function in zebrafish development and in mammalian cells, we revealed a conserved role for minor splicing in cell-cycle progression. We argue that the segregation of the splicing systems allows for processing of partially unspliced cytoplasmic transcripts, emerging as a result of different splicing rates. The segregation offers a mechanism accounting for spliceosome evolution in a single lineage and provides a means for nucleus-independent control of gene expression.

Adenovirus E1A negatively and positively modulates transcription of AP-1 dependent genes by dimer-specific regulation of the DNA binding and transactivation activities of Jun

Adenovirus E1A proteins inhibit expression of the collagenase gene but activate expression of the c‐jun gene. Both effects are mediated by TPA‐responsive elements (TREs), the binding sites for members of the AP‐1 transcription factor family. By a process that is independent of the retinoblastoma gene product, E1A distinguishes between different AP‐1 factors: in vivo binding of Jun/Jun homodimers and Jun/Fos heterodimers to the collagenase TRE is totally blocked by E1A while, in contrast, there is no inhibition of Jun/ATF‐2 binding to the TRE sequences in the c‐jun promoter. Altered phosphorylation of the DNA binding domain of cJun is not involved in the inhibition of cJun/cJun and cJun/cFos binding. E1A does, however, cause hyperphosphorylation of the transactivation domain of cJun, which is likely to be responsible for the enhanced c‐jun transcription by E1A mediated through cJun/ATF‐2 heterodimers.

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.