The human caspase-2 gene: alternative promoters, pre-mRNA splicing and AUG usage direct isoform-specific expression Emmanuelle Logette, Anne Wotawa, Ste´phanie Solier, Lydie Desoche, Eric Solary and Laurent Corcos* Inserm U517, Faculty of Medicine, 7 Bd Jeanne d’Arc, 21000 Dijon, France Caspases have been shown to play important roles in apoptotic cell death, cytokine maturation and cell differentiation. However, the transcriptional regulation of the corresponding CASP genes remains poorly known. We describe a 5.1 kb fragment located upstream of the first translated exon in the human CASP-2 gene, which is known to encode caspase-2L and -2S protein isoforms. Transient transfection experiments, together with tran- scription start site mapping and transcript analysis, demonstrate that each caspase mRNA is initiated from separate promoter regions, and produced from alternative splicing events in these regions. The CASP-2L promoter is much stronger than the CASP-2S promoter, in good agreement with the respective transcript levels of the two caspases. In addition, several in-frame translational start sites can be identified for each isoform, one of which is common to both, present in the second common exon, and used efficiently. Surprisingly, the short isoform may also be initiated at a downstream AUG codon within the same exon. Thus, promoter strength, alternative transcriptional initiation and 5 0 -splicing events regulate the expression of the main caspase-2 isoforms that may be translated from alternative translation initiation codons. Oncogene (2003) 22, 935–946. doi:10.1038/sj.onc.1206172 Keywords: caspase-2 gene; casp-2S; casp-2L; AUG; mRNA splicing; transcriptional regulation Introduction A family of cysteine proteases, known as caspases, plays an important role in many forms of cell death by apoptosis and proinflammatory cytokine maturation (Thornberry and Lazebnik, 1998). So far, 14 caspases have been identified in mammalian cells, of which 12 human enzymes are known (Nicholson, 1999). The second identified mammalian caspase, known as cas- pase-2 (Alnemri et al., 1996), was initially referred to as Nedd-2 in mice (Kumar et al., 1992) and Ich-1 (interleukin-lb converting enzyme homolog 1) in humans (Wang et al., 1994). Nedd-2 was originally identified as a developmentally regulated gene in mouse brain (Kumar et al., 1992). The human CASP-2 gene, Ich-1, encodes at least two different forms of mRNA species derived from alternative splicing in the 3 0 region (Wang et al., 1994). Overexpression of the long isoform caspase-2L was shown to induce programmed cell death (Kumar et al., 1994), whereas its decreased expression by antisense technology delayed apoptosis in response to various stimuli (Kumar, 1995; Troy et al., 2000; Droin et al., 2001a). Conversely, overexpression of the short isoform, caspase-2S, could suppress mammalian cell death (Wang et al., 1994; Kumar, 1995; Droin et al., 2001b). Experiments using caspase-2-knockout mice showed that oocytes were resistant to cell death induced by cytotoxic drugs and that B lymphoblasts were resistant to perforin- and granzyme B-induced apoptosis whereas cell death was accelerated in some neuronal populations (Bergeron et al., 1998; Morita et al., 2001). Altogether, these observations indicate that caspase-2 may act both as a positive and a negative regulator of programmed cell death, depending on cell lineage, development stage and stimulus. Inclusion or skipping of a 61-base pair (bp) exon in the 3 0 -end of casp-2 pre-mRNA leads to the forma- tion of two mRNAs encoding the short and the long isoform of caspase-2, respectively (Wang et al., 1994; Cote et al., 2001a). Insertion of the alternative exon 9 introduces a premature stop codon, which leads to production of caspase-2S (Wang et al., 1994; Cote et al., 2001b). In addition, the two mRNAs differ at their 5 0 - end, upstream of the first exon common to both isoforms, which suggests the existence of distinct transcriptional initiation sites and/or alternative splicing events in this region. The initial survey of CASP-2 gene expression showed that the long isoform is the dominant form expressed in most tissues examined whereas the short isoform is predominantly expressed in heart, brain and skeletal muscle (Wang et al., 1994; Kumar et al., 1997). In the course of rat brain development, casp-2S mRNA reaches similar amounts as casp-2L mRNA, and remains at this level in the adult tissue (Kojima et al., 1998). Additional caspase-2 isoforms may also be derived from CASP-2 gene alternative splicing. For example, we have identified a truncated isoform, casp-2L-Pro, whose overexpression Received 10 July 2002; revised 1 October 2002; accepted 22 October 2002 *Correspondence: L Corcos; E-mail: laurent.corcos@u-bourgogne.fr Oncogene (2003) 22, 935–946 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc ONCOGENOMICS