Identification of a novel splice variant of X-linked inhibitor of apoptosis-associated factor 1 Weihong Yin, Satish Cheepala, John L. Clifford * Department of Biochemistry and Molecular Biology, Louisiana State University Health Science Center School of Medicine in Shreveport and Feist-Weiller Cancer Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130, USA Received 8 November 2005 Available online 5 December 2005 Abstract XAF1 (XIAP-associated factor 1) binds to XIAP and blocks its anti-apoptotic activity. It has been reported that XAF1 is mainly expressed in normal tissues but is missing or present at low levels in most cancer cell lines, which implies a tumor-suppressing function. In the present study we describe the identification of a novel splice variant of human XAF1, designated XAF1C, which contains a cryptic exon. Incorporation of this exon (exon 4b) into the mRNA introduces an in-frame stop codon, resulting in a shortened open-reading frame (ORF) of 495 nucleotides. This ORF is predicted to encode a 164 amino acid (AA) protein lacking the C-terminal domain of the previously described XAF1(A), but containing a unique 24 AA carboxy terminus. Like XAF1(A), XAF1C mRNA expression was detected in a variety of human cancer cell lines and also in normal human tissues. The ratio of XAF1(A) and XAF1C mRNA expres- sion differs amongst the cell lines tested, suggesting differential mRNA stabilities and/or the existence of tissue- or cell type-specific splic- ing regulation. In transfected cells, xaf1c encodes a truncated protein of 18 kDa, which is distributed primarily in the nucleus. Ó 2005 Elsevier Inc. All rights reserved. Keywords: XIAP; XAF1; Splice variant; Apoptosis Apoptosis, or programmed cell death, plays an impor- tant role in many physiological processes, especially in the immune system [1,2], nervous system [3,4], and in devel- opment [5,6]. Deregulated apoptosis is associated with the pathogenesis of many human diseases, such as AIDS, auto- immunity, and cancer [7–9]. Apoptotic cell death is facili- tated by a set of cysteine proteases known as ÔcaspasesÕ (for Cysteine Aspartyl-specific Proteases) [10,11], which exert their biological effects by cleaving diverse cellular sub- strates that are essential for normal cell functions. Caspase activity can be negatively regulated by a class of endoge- nous proteins, the IAP (inhibitors of apoptosis protein) family proteins, which have one or more baculoviral IAP repeat (BIR) domains. In humans, at least nine IAP family members have been identified (XIAP, cIAP-1, cIAP-2, NAIP, ML-IAP, ILP2, KIAP, apollon, and survivin). Among these, X-linked IAP (XIAP) is the most potent cas- pase inhibitor [12,13]. A novel protein has recently been identified and termed XIAP-associated factor 1 (XAF1), due to its ability to bind and directly interact with XIAP [14]. XAF1 is a 301-amino acid protein containing six zinc finger motifs at its N-termi- nus. Structural analysis indicated that the C-terminal por- tion of XAF1 is critical for its pro-apoptotic function while the N-terminal zinc-finger region is required for self-associ- ation or interaction with other proteins [15]. In vitro stud- ies revealed that XAF1 could block the XIAP-mediated inhibition of caspase-3 activity and depletion of XAF1 by anti-sense RNA resulted in increased resistance to etopo- side-triggered apoptosis [16]. Over-expression of XAF1 in HeLa cells leads to the redistribution of XIAP from the cytosol to the nucleus [16] and enhances TNF-related apoptosis-inducing ligand (TRAIL)-initiated apoptosis in melanoma cells [15]. Interferon treatment sensitizes several 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.128 * Corresponding author. Fax: +1 318 675 5180. E-mail address: jcliff@lsuhsc.edu (J.L. Clifford). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 339 (2006) 1148–1154 BBRC