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Regulation of Apoptosis by Alternative Pre-mRNA Splicing

2005; Elsevier BV; Volume: 19; Issue: 1 Linguagem: Inglês

10.1016/j.molcel.2005.05.026

1097-4164

Christian Schwerk, Klaus Schulze‐Osthoff,

RNA and protein synthesis mechanisms

Apoptosis, a phenomenon that allows the regulated destruction and disposal of damaged or unwanted cells, is common to many cellular processes in multicellular organisms. In humans more than 200 proteins are involved in apoptosis, many of which are dysregulated or defective in human diseases including cancer. A large number of apoptotic factors are regulated via alternative splicing, a process that allows for the production of discrete protein isoforms with often distinct functions from a common mRNA precursor. The abundance of apoptosis genes that are alternatively spliced and the often antagonistic roles of the generated protein isoforms strongly imply that alternative splicing is a crucial mechanism for regulating life and death decisions. Importantly, modulation of isoform production of cell death proteins via pharmaceutical manipulation of alternative splicing may open up new therapeutic avenues for the treatment of disease. Apoptosis, a phenomenon that allows the regulated destruction and disposal of damaged or unwanted cells, is common to many cellular processes in multicellular organisms. In humans more than 200 proteins are involved in apoptosis, many of which are dysregulated or defective in human diseases including cancer. A large number of apoptotic factors are regulated via alternative splicing, a process that allows for the production of discrete protein isoforms with often distinct functions from a common mRNA precursor. The abundance of apoptosis genes that are alternatively spliced and the often antagonistic roles of the generated protein isoforms strongly imply that alternative splicing is a crucial mechanism for regulating life and death decisions. Importantly, modulation of isoform production of cell death proteins via pharmaceutical manipulation of alternative splicing may open up new therapeutic avenues for the treatment of disease. Apoptosis or programmed cell death can be triggered by a multitude of different stimuli but is mainly accomplished by either one of two major pathways (Adams, 2003Adams J.M. Ways of dying: multiple pathways to apoptosis.Genes Dev. 2003; 17: 2481-2495Crossref PubMed Scopus (659) Google Scholar). Whereas the extrinsic pathway involves the transmission of extracellular signals to the intracellular death machinery via the binding of signaling molecules to and the subsequent activation of cell surface receptors (Figure 1A ), the intrinsic pathway requires the release of proapoptotic factors from the mitochondria via the disruption of the outer mitochondrial membrane barrier function (Figure 1B). Both pathways converge onto a common executive cell-death machinery, which is largely conserved between different organisms. Required for regulation and execution of apoptosis is an extended set of factors, which can be divided into different families dependent on their domain structure and function during cell death (Aravind et al., 2001Aravind L. Dixit V.M. Koonin E.V. Apoptotic molecular machinery: vastly increased complexity in vertebrates revealed by genome comparisons.Science. 2001; 291: 1279-1284Crossref PubMed Scopus (282) Google Scholar, Reed et al., 2004Reed, J.C., Doctor, K.S., and Godzik, A. (2004). The domains of apoptosis: a genomics perspective. Sci. STKE, RE9. 10.1126/stke.2392004re9.Google Scholar). Initially, genetic analyses in the nematode C. elegans had revealed three proteins, termed CED-3, CED-4, and CED-9, each of which belongs to a distinct family of apoptotic factors (Horvitz et al., 1994Horvitz H.R. Shaham S. Hengartner M.O. The genetics of programmed cell death in the nematode Caenorhabditis elegans.Cold Spring Harb. Symp. Quant. Biol. 1994; 59: 377-385Crossref PubMed Scopus (168) Google Scholar). Sequence comparisons lead to the conclusion that CED-3 is a homolog of the mammalian interleukin-1β (IL-1β)-converting enzyme (ICE), a member of the family of cysteinyl aspartate-specific proteases, which are now termed caspases (Los et al., 1999Los M. Wesselborg S. Schulze-Osthoff K. The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice.Immunity. 1999; 10: 629-639Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Although ICE belongs to the group of inflammatory caspases and is not involved in programmed cell death, several members of the caspase family are proteolytically activated during apoptosis and play key roles during the execution of apoptosis via cleavage of selected target proteins. Apoptotic caspases can be distinguished into two groups: the initiator caspases (caspase-2, -8, -9, and -10), which are mainly required for the processing and activation of other caspases, and the effector caspases (caspase-3, -6, and -7), which are the executioners of apoptosis and cleave specific target proteins (Earnshaw et al., 1999Earnshaw W.C. Martins L.M. Kaufmann S.H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis.Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2395) Google Scholar). The vertebrate homolog of CED-4 is the apoptotic protease-activating factor-1 (Apaf-1), an adaptor protein that functions together with cytochrome c to mediate activation of caspases during the mitochondrial pathway of apoptosis via formation of the apoptosome, a multiprotein caspase-activating complex (Figure 1B) (Adams, 2003Adams J.M. Ways of dying: multiple pathways to apoptosis.Genes Dev. 2003; 17: 2481-2495Crossref PubMed Scopus (659) Google Scholar). Specific domains with a similar secondary structure characterize Apaf-1 as well as other adaptor proteins. These domains include the caspase recruitment domain (CARD), death domain (DD), death effector domain (DED), Toll/IL-1R homology domain (TIR), and the baculoviral inhibitor of apoptosis protein (IAP) repeat (BIR) motif that serve to connect the upper signaling apoptotic pathways with the downstream machinery (Reed et al., 2004Reed, J.C., Doctor, K.S., and Godzik, A. (2004). The domains of apoptosis: a genomics perspective. Sci. STKE, RE9. 10.1126/stke.2392004re9.Google Scholar). In C. elegans the activity of CED-4 is controlled via interaction with the antiapoptotic cell death protein CED-9. Mammalian homologs of CED-9 turned out to belong to the Bcl-2 family of apoptotic regulators. These proteins are characterized by the presence of up to four conserved α-helical sequences, the so-called Bcl-2 homology (BH) domains. The highly conserved antiapoptotic Bcl-2 proteins possess all four domains. In this group belong CED-9 and the mammalian counterparts Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1. The proapoptotic Bcl-2 family members can be divided into two groups. The first comprises the more-conserved multidomain proapoptotic proteins Bax, Bak, and Bok, which contain BH1, BH2, and BH3 domains. The second group includes the mammalian BH3-only proteins (Bid, Bad, Bim, Bik, Noxa, and Puma) as well as C. elegans EGL-1. The function of Bcl-2 proteins during apoptosis in mammals is complex and involves activation and translocation of proapoptotic members to the outer mitochondrial membrane followed by membrane perturbation and release of apoptogenic factors including cytochrome c (Adams, 2003Adams J.M. Ways of dying: multiple pathways to apoptosis.Genes Dev. 2003; 17: 2481-2495Crossref PubMed Scopus (659) Google Scholar, Green and Kroemer, 2004Green D.R. Kroemer G. The pathophysiology of mitochondrial cell death.Science. 2004; 305: 626-629Crossref PubMed Scopus (2696) Google Scholar). It is important to note that a major increase in complexity documented in the number of the proteins involved in cell death as well as their apoptotic domains took place during vertebrate evolution. This is exemplified by another family of apoptotic proteins, the death receptors and their ligands, which trigger the extrinsic pathway of apoptosis (Figure 1A). In vertebrates cell death can be induced directly by specific death ligands, which belong to the tumor necrosis factor (TNF) family and bind to the death receptors. These transmembrane receptors constitute a subgroup of the TNF receptor superfamily, which includes the Fas receptor (CD95), TNFR1, and receptors for the TNF-related apoptosis-inducing ligand (TRAIL). Death receptors are characterized by the presence of an intracellular death domain (DD) that is required for the transmission of the apoptotic signal via the recruitment of DD-containing adaptor molecules. The adaptor proteins contain further specific domains (e.g., the DED or the CARD) that enable interaction with downstream apoptotic molecules. Other TNFR-interacting proteins comprise the family of the TNFR-associated factors (TRAFs), which connect receptors with downstream factors including the NF-κB and JNK signaling pathways (Schulze-Osthoff et al., 1998Schulze-Osthoff K. Ferrari D. Los M. Wesselborg S. Peter M.E. Apoptosis signaling by death receptors.Eur. J. Biochem. 1998; 254: 439-459Crossref PubMed Scopus (844) Google Scholar, Aggarwal, 2003Aggarwal B.B. Signaling pathways of the TNF superfamily: a double-edged sword.Nat. Rev. Immunol. 2003; 3: 745-756Crossref PubMed Scopus (2018) Google Scholar). Due to this extended functional diversity and the huge number of factors involved, apoptosis needs to be tightly regulated by a finely organized genetic program. It would be beyond the scope of this review to summarize all factors involved in transcriptional regulation of the apoptotic program, but we would like to point out the outstanding role of the p53 family of transcriptional regulators in development and apoptosis (Melino et al., 2002Melino G. De Laurenzi V. Vousden K.H. p73: friend or foe in tumorigenesis.Nat. Rev. Cancer. 2002; 2: 605-615Crossref PubMed Scopus (492) Google Scholar, Vousden and Lu, 2002Vousden K.H. Lu X. Live or let die: the cell's response to p53.Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2620) Google Scholar). In addition to transcriptional regulation, control of programmed cell death can occur on multiple other levels, including the regulation of pre-mRNA processing by alternative splicing. Concomitant with the evolution of higher eukaryotes, cells were required to raise their protein diversity in order to cope with the increasingly broad spectrum of functional and behavioral complexity. One of the major approaches to accomplish this task is the generation of multiple transcript species from a common mRNA precursor. This phenomenon, which is termed alternative splicing, adds an additional layer of control to the gene expression process because it leads to the production of discrete protein isoforms, which can have distinct functions in cellular events. Alternative splicing has been shown to be relevant for such diverse processes as apoptosis, sex determination, axon guidance, and cell excitation and contraction (Maniatis and Tasic, 2002Maniatis T. Tasic B. Alternative pre-mRNA splicing and proteome expansion in metazoans.Nature. 2002; 418: 236-243Crossref PubMed Scopus (587) Google Scholar, Black, 2003Black D.L. Mechanisms of alternative pre-messenger RNA splicing.Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1885) Google Scholar). In human cells the protein-coding parts of genes, so-called exons, are interrupted, on average, by 10-fold longer noncoding introns. Although the introns are transcribed by RNA polymerase II, they have to be removed from the precursor to obtain the mature transcript. In higher eukaryotes most genes consist of several exons and introns, and alternative splicing allows the removal and joining of selected parts of the precursor transcript. It is estimated that about 60% of human gene products are subject to this kind of regulation (Modrek and Lee, 2003Modrek B. Lee C.J. Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss.Nat. Genet. 2003; 34: 177-180Crossref PubMed Scopus (418) Google Scholar). The alternative splicing patterns of pre-mRNAs can be rather complicated with up to thousands of products. This astonishing complexity requires the process of intron removal to be tightly controlled and regulated. Not surprisingly, disruption of normal splicing patterns can lead to disease in humans (Faustino and Cooper, 2003Faustino N.A. Cooper T.A. Pre-mRNA splicing and human disease.Genes Dev. 2003; 17: 419-437Crossref PubMed Scopus (945) Google Scholar). Removal of the introns from pre-mRNA molecules and the joining of the exons are guided by nucleotide sequences at the intron-exon junctions called splice sites (Figure 2). Located at the 5′ end of the intron is the 5′ splice site, which is distinguished by a GU dinucleotide at the intron end (encompassed within a larger, less-conserved consensus sequence). The 3′ splice site at the 3′ end of the intron displays three conserved nucleotide sequences. These are the branch point sequence followed by a polypyrimidine tract and an AG at the 3′ terminus of the intron (Brow, 2002Brow D.A. Allosteric cascade of spliceosome activation.Annu. Rev. Genet. 2002; 36: 333-360Crossref PubMed Scopus (286) Google Scholar). Splicing of pre-mRNA molecules is accomplished by the spliceosome, a macromolecular machinery composed of various small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP protein components. The spliceosome is assembled in a stepwise fashion and undergoes several alterations and conformational changes during the splicing process. This process starts with the binding of the U1 snRNP to the 5′ splice site, followed by the association of the U2 snRNP with the branch point, a step that is guided by the U2 auxiliary factor (U2AF) bound to the 3′ splice site. Subsequently, U4, U5, and U6 snRNPs join the growing splicing machinery. U1 and U4 RNAs exit the spliceosome before it becomes catalytically active, a process that requires extensive remodeling of the splicing machinery (Staley and Guthrie, 1998Staley J.P. Guthrie C. Mechanical devices of the spliceosome: motors, clocks, springs, and things.Cell. 1998; 92: 315-326Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar, Brow, 2002Brow D.A. Allosteric cascade of spliceosome activation.Annu. Rev. Genet. 2002; 36: 333-360Crossref PubMed Scopus (286) Google Scholar). During spliceosome formation at a certain splice site, the splice site consensus sequences are helped by nonsplice site regulatory sequences that strongly affect spliceosome assembly (Figure 2). These regulators can work as enhancers as well as silencers of splicing. The regulatory sequences can be located in exons and are called exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs), respectively. Also, intronic splicing enhancers (ISEs) and silencers (ISSs) exist (Black, 2003Black D.L. Mechanisms of alternative pre-messenger RNA splicing.Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1885) Google Scholar). Most important for an error-free splicing process is the proper recognition of splice site sequences and association between 5′ and 3′ splice sites. To fulfill this task, the basic splicing machinery is assisted by a large number of non-snRNP protein components (Figure 2), most notably members of the heteronuclear ribonucleoprotein family (hnRNP proteins) and proteins containing serine-arginine-rich sequences (SR proteins). These factors, together with various other RNA binding proteins, are also substantial in regulating alternative splicing by modulating spliceosome assembly and splice site choice. In this process, SR and hnRNP proteins may play an antagonistic role, with SR proteins being positive regulators and hnRNP proteins negative regulators of RNA processing (Manley and Tacke, 1996Manley J.L. Tacke R. SR proteins and splicing control.Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (597) Google Scholar, Graveley, 2000Graveley B.R. Sorting out the complexity of SR protein functions.RNA. 2000; 6: 1197-1211Crossref PubMed Scopus (862) Google Scholar). From the above it is clear that alternative splicing can serve to fine-tune important and complex cellular events. There is increasing evidence that this mode of regulation of gene expression also plays a major role in the control of programmed cell death (Wu et al., 2003Wu J.Y. Tang H. Havlioglu N. Alternative pre-mRNA splicing and regulation of programmed cell death.Prog. Mol. Subcell. Biol. 2003; 31: 153-185Crossref PubMed Scopus (53) Google Scholar). As will be summarized below, expression of a huge amount of proteins directly involved in the apoptotic pathways is subject to regulation by alternative splicing. Proteins belonging to each family of cell death factors are alternatively spliced, and in many cases the different isoforms produced have distinct and often even opposing functions during programmed cell death. A schematic overview emphasizing the diversity of apoptotic factors and pathways targeted by this kind of regulation is given in Figure 3. It should be mentioned that in many cases alternative splice variants have been described only at the RNA level. Alternative splicing can also regulate gene expression by splicing transcripts into unproductive mRNAs that are targeted to the nonsense-mediated decay pathway. In this case, some of the mRNA transcripts do not encode a functional polypeptide but, rather, function to prevent overexpression of the main protein. Therefore, alternatively spliced transcripts might often only be weakly expressed, making an analysis of their physiological function difficult. In the following sections we will focus on the more extensively analyzed and most interesting apoptotic factors that are subject to alternative splicing (summarized in Table 1). A more comprehensive overview of alternatively spliced proteins directly involved in the extrinsic and intrinsic death pathways is given in Table S1 in the Supplemental Data available with this article online.Table 1Examples of Alternatively Spliced Apoptotic Factors and Functional ConsequencesaFor a more comprehensive overview of alternatively spliced apoptotic factors involved in the extrinsic and intrinsic death pathways, please refer to Table S1.ProteinCellular FunctionFunctional Consequences of Alternative SplicingLigands and ReceptorsFasLactivation of death receptor-mediated apoptosisaltered solubility and apoptotic potentialFasapoptosis, extrinsic pathwayaltered solubility; dominant-negative phenotypeLARDapoptosis, extrinsic pathway (lymphocytes)altered solubility and apoptotic potentialAdaptor Proteins and RegulatorsTRAF2TNF receptor signalingdominant-negative phenotypeTRAF3TNF receptor signalingdistinct functions in signal transductionMyDD88Toll-like receptor signalingdominant-negative phenotypeMADDactivation of MAPKantagonistic functions during apoptosisApaf-1component of the apoptosomeantagonistic functions during apoptosissurvivinregulation of apoptosis and cell cyclechanged cellular localization and apoptotic potentialSmac/Diablomitochondrial IAP-binding proteinaltered apoptotic potentialBcl-2 FamilyBcl-xantiapoptoticantagonistic functions during apoptosisBakproapoptoticaltered apoptotic potential (cell type specific)Bidconnection between extrinsic and intrinsic apoptotic pathwaysaltered apoptotic potentialBimproapoptotic (BH3-only)changed cellular localization (microtubule binding)Caspases and Caspase-like Proteinscaspase-2initiator caspase, substrate cleavageantagonistic functions during apoptosiscaspase-9initiator caspase, substrate cleavagedominant-negative phenotypecaspase-10initiator caspase, substrate cleavagechanged activityFLIPregulation of death-receptor mediated apoptosispotential antagonistic functions during immune responseCaspase TargetsICADapoptotic DNA fragmentationchanged cellular distributionCADapoptotic DNA fragmentationAcinusapoptotic chromatin condensation; RNA splicingp53 Familyp53, p63, and p73regulation of apoptosis and cell cyclealtered apoptotic potentiala For a more comprehensive overview of alternatively spliced apoptotic factors involved in the extrinsic and intrinsic death pathways, please refer to Table S1. Open table in a new tab Binding of specialized ligands of the TNF family to their respective receptors initiates the extrinsic pathway of apoptosis. Death ligands are usually expressed as type II transmembrane proteins, but most of them also exist in a soluble form generated by proteolytic cleavage of the extracellular domain by rather specific metalloproteases. Proteolysis can have a significant impact on the apoptotic potential as exemplified by soluble variants of FasL, which can block the death-promoting activity of the membrane bound variant (Schulze-Osthoff et al., 1998Schulze-Osthoff K. Ferrari D. Los M. Wesselborg S. Peter M.E. Apoptosis signaling by death receptors.Eur. J. Biochem. 1998; 254: 439-459Crossref PubMed Scopus (844) Google Scholar, Aggarwal, 2003Aggarwal B.B. Signaling pathways of the TNF superfamily: a double-edged sword.Nat. Rev. Immunol. 2003; 3: 745-756Crossref PubMed Scopus (2018) Google Scholar). A soluble isoform of FasL can also be generated by alternative splicing, as has been described for murine FasL. This FasL isoform lacks the transmembrane, the intracellular domain, and part of the extracellular domain and, similarly to proteolytically derived soluble FasL, inhibits apoptosis via the Fas receptor pathway (Ayroldi et al., 1999Ayroldi E. D'Adamio F. Zollo O. Agostini M. Moraca R. Cannarile L. Migliorati G. Delfino D.V. Riccardi C. Cloning and expression of a short Fas ligand: a new alternatively spliced product of the mouse Fas ligand gene.Blood. 1999; 94: 3456-3467Crossref PubMed Google Scholar). Death receptor-mediated cell death is also regulated by the production of distinct receptor isoforms with specific functions during apoptosis. In both activated human peripheral blood mononuclear cells and T cell tumor lines, several soluble variants of Fas generated by alternative splicing and lacking the transmembrane domain have been described (Cascino et al., 1995Cascino I. Fiucci G. Papoff G. Ruberti G. Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing.J. Immunol. 1995; 154: 2706-2713PubMed Google Scholar, Hughes and Crispe, 1995Hughes D.P. Crispe I.N. A naturally occurring soluble isoform of murine Fas generated by alternative splicing.J. Exp. Med. 1995; 182: 1395-1401Crossref PubMed Scopus (87) Google Scholar). Among these is FasExo6Del, which misses the transmembrane domain due to alternative splicing of the intact exon 6. FasExo6Del can compete with the membrane bound form of Fas and functions as a soluble decoy (Cheng et al., 1994Cheng J. Zhou T. Liu C. Shapiro J.P. Brauer M.J. Kiefer M.C. Barr P.J. Mountz J.D. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule.Science. 1994; 263: 1759-1762Crossref PubMed Scopus (1083) Google Scholar, Cascino et al., 1995Cascino I. Fiucci G. Papoff G. Ruberti G. Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing.J. Immunol. 1995; 154: 2706-2713PubMed Google Scholar). Another splice variant of Fas, termed FasExo8Del, lacks the intracellular DD due to a reading frame change caused by the skipping of exon 8. FasExo8Del was identified in a human lymphoma cell line resistant to Fas-mediated apoptosis and found to be responsible for the resistant phenotype in a dominant-negative fashion (Cascino et al., 1996Cascino I. Papoff G. De Maria R. Testi R. Ruberti G. Fas/Apo-1 (CD95) receptor lacking the intracytoplasmic signaling domain protects tumor cells from Fas-mediated apoptosis.J. Immunol. 1996; 156: 13-17PubMed Google Scholar). Among other functions the Fas-FasL system is important for regulation of the immune system in several ways, including negative selection and activation-induced cell death in lymphocytes (Schulze-Osthoff et al., 1998Schulze-Osthoff K. Ferrari D. Los M. Wesselborg S. Peter M.E. Apoptosis signaling by death receptors.Eur. J. Biochem. 1998; 254: 439-459Crossref PubMed Scopus (844) Google Scholar, Aggarwal, 2003Aggarwal B.B. Signaling pathways of the TNF superfamily: a double-edged sword.Nat. Rev. Immunol. 2003; 3: 745-756Crossref PubMed Scopus (2018) Google Scholar). The production of soluble FasL by alternative splicing is upregulated in T cells after T cell receptor activation, supporting a regulatory role of splice site selection (Ayroldi et al., 1999Ayroldi E. D'Adamio F. Zollo O. Agostini M. Moraca R. Cannarile L. Migliorati G. Delfino D.V. Riccardi C. Cloning and expression of a short Fas ligand: a new alternatively spliced product of the mouse Fas ligand gene.Blood. 1999; 94: 3456-3467Crossref PubMed Google Scholar). Along this line, the lymphocyte-associated receptor of death (LARD, TNFRSF25) is expressed in at least 11 different isoforms, with membrane bound and soluble variants, distinguished by the presence or absence of a transmembrane domain. Production of LARD isoforms is thought to be controlled via a programmed change in alternative splicing during T cell activation, indicating an involvement in the control of lymphocyte proliferation (Screaton et al., 1997Screaton G.R. Xu X.N. Olsen A.L. Cowper A.E. Tan R. McMichael A.J. Bell J.I. LARD: a new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing.Proc. Natl. Acad. Sci. USA. 1997; 94: 4615-4619Crossref PubMed Scopus (178) Google Scholar). Taken together, increasing evidence points to a role of alternatively spliced death receptor components for the regulation of T cell homeostasis. The relevance of alternative splicing for death receptor systems is further highlighted by the observation that elevated levels of soluble Fas can be detected in patients with systemic lupus erythematosus and other malignancies. Furthermore, expression of soluble FasL is associated with several diseases, including cancers and immunological ailments (Cheng et al., 1994Cheng J. Zhou T. Liu C. Shapiro J.P. Brauer M.J. Kiefer M.C. Barr P.J. Mountz J.D. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule.Science. 1994; 263: 1759-1762Crossref PubMed Scopus (1083) Google Scholar, Janssen et al., 2003Janssen O. Qian J. Linkermann A. Kabelitz D. CD95 ligand–death factor and costimulatory molecule?.Cell Death Differ. 2003; 10: 1215-1225Crossref PubMed Scopus (69) Google Scholar), suggesting that detection of soluble Fas and FasL could serve as a potential marker in these diseases. TNFR-associated factors (TRAFs) are adaptor proteins required to connect TNFRs with downstream signaling pathways. So far six mammalian TRAFs have been identified, some of which are alternatively spliced. Two different isoforms of TRAF2 have been described. Whereas TRAF2 mediates activation of NF-κB, TRAF2A, which contains a seven amino acid insertion into the RING finger domain, can act as a dominant-negative inhibitor of NF-κB (Brink and Lodish, 1998Brink R. Lodish H.F. Tumor necrosis factor receptor (TNFR)-associated factor 2A (TRAF2A), a TRAF2 splice variant with an extended RING finger domain that inhibits TNFR2-mediated NF-kappaB activation.J. Biol. Chem. 1998; 273: 4129-4134Crossref PubMed Scopus (57) Google Scholar). Similarly, the TRAF3 gene encodes several different splice variants that display distinct functions in signal transduction (van Eyndhoven et al., 1999van Eyndhoven W.G. Gamper C.J. Cho E. Mackus W.J. Lederman S. TRAF-3 mRNA splice-deletion variants encode isoforms that induce NF-kappaB activation.Mol. Immunol. 1999; 36: 647-658Crossref PubMed Scopus (29) Google Scholar). TRAF6 as well as the DD-containing adaptor protein MyD88 are required for efficient signaling by members of the interleukin-1 receptor (IL-1R) family, including the Toll-like receptors (TLRs) that are involved in early host defense against invading pathogens (Akira and Takeda, 2004Akira S. Takeda K. Toll-like receptor signaling.Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6343) Google Scholar). MyD88 contains a C-terminal Toll and IL-1R homology (TIR) domain and an N-terminal DD for interaction with the TLR and IL-1R as well as the IL-1R-associated kinases (IRAKs), respectively. Binding to MyD88 triggers phosphorylation of IRAK1 via IRAK4. Phosphorylated IRAK1 in turn associates with TRAF6 and mediates activation of NF-κB. Two different isoforms of MyD88 (termed MyD88L and MyD88S) are generated by alternative splicing, and MyD88S lacks a small intermediate domain between the DD and the TIR domain. MyD88S protein expression can be detected under conditions of chronic inflammation or after exposure of macrophages to LPS (Janssens et al., 2002Janssens S. Burns K. Tschopp J. Beyaert R. Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88.Curr. Biol. 2002; 12: 467-471Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Interestingly, MyD88S functions as a dominant-negative inhibitor of IL-1- and LPS-induced, but not TNF-induced, NF-κB activation. This behavior is due to the inability of MyD88S to recruit IRAK4, which is functional in phosphorylation and activation of IRAK1 (Burns et al., 2003Burns K. Janssens S. Brissoni B. Olivos N. Beyaert R. Tschopp J. Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4.J. Exp. Med. 2003; 197: 263-268Crossref PubMed Scopus (392) Google Scholar). The CED-4 homolog Apaf-1 functions as an adaptor protein during execution of the mitochondrial pathway of apoptosis. Inclusion of an additional C-terminal WD-40 repeat in human Apaf-1 via alternative splicing seems to be required for a proapoptotic function (Benedict et al., 2000Benedict M.A. Hu Y. Inohara N. Nunez G. Expression and functional analysis of Apa