Molecular cloning of novel alternatively spliced variants of BCL2L12, a new member of the BCL2 gene family, and their expression analysis in cancer cells Christos K. Kontos, Andreas Scorilas ⁎ Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens, Greece abstract article info Article history: Accepted 27 April 2012 Available online 1 June 2012 Keywords: Apoptosis Alternative splicing Novel splice variants EST clones Non-sense mediated mRNA decay (NMD) BH3-only proteins In the past, we identified and cloned the BCL2-like 12 (BCL2L12) gene, a novel member of the BCL2 family, which is implicated in various malignancies. The classical BCL2L12 protein isoform contains a highly con- served BH2 domain, a BH3-like motif, and a proline-rich region, and is involved in apoptosis. Most members of this apoptosis-related family are subjected to alternative splicing, thus generating multiple protein isoforms with distinct properties, and sometimes even with opposite function (pro- vs. anti-apoptotic). In the current study, we report the identification, molecular cloning, and expression pattern of novel splice var- iants of the human BCL2L12 gene in cancer cell lines. EST clones displaying high sequence identity (≥90%) with the classical BCL2L12 transcript were aligned, in order to identify those containing at least one novel splice junction. EST database mining led to the identification of three previously unknown splice variants of this apoptotic gene. In our effort to experimentally validate these novel transcripts, we also cloned seven more, previously unidentified, BCL2L12 alternatively spliced variants. Expression analysis of all BCL2L12 splice variants in human cancer cell lines and embryonic kidney cells revealed remarkable differ- ences between their BCL2L12 expression profiles. Interestingly, 7 out of 10 novel splice variants of BCL2L12 are predicted to encode new protein isoforms, some of which are BH3-only proteins, in contrast to the clas- sical BCL2L12 isoform, which also contains a functional BH2 domain. The remaining three novel splice vari- ants of BCL2L12 are nonsense-mediated mRNA decay (NMD) candidates. © 2012 Elsevier B.V. All rights reserved. 1. Introduction BCL2L12 is a newly identified member of the BCL2 family of apoptosis-related genes. Currently, three distinct transcripts resulting from alternative splicing of the BCL2L12 gene are known. The largest splice variant (BCL2L12 v.1) consists of seven coding exons and its translation produces the classical BCL2L12 protein isoform (BCL2L12 is.1), a 334-amino acid (aa) polypeptide containing a highly con- served BH2 domain, a BH3-like motif, and a proline-rich region (Kontos et al., 2009; Scorilas et al., 2001). Expression of the full- length mRNA transcript has been observed in many tissues, including breast, thymus, prostate, fetal liver, colon, placenta, pancreas, small intestine, spinal cord, kidney, and bone marrow. An alternative splice variant lacking exon 3 and designated as BCL2L12-A (or BCL2L12 v.2) is mainly expressed in fetal liver, spinal cord, and skeletal muscle (Scorilas et al., 2001). Furthermore, the sequence of a third BCL2L12 splice variant (BCL2L12 v.3) that makes use of an alternate in-frame splice site at the 5′ end of exon 3, compared to the full-length tran- script, has been deposited in GenBank. The resulting isoform (BCL2L12 is.3) has the same N- and C-termini compared to the main isoform, but is shorter by 1 aa (Thomadaki and Scorilas, 2006). Data about the localization of the BCL2L12 protein seem to be con- fusing at the moment. Initially, this protein was detected both in cy- tosol and mitochondria (Toumelin et al., 2006), yet Stegh et al. (2007) reported that BCL2L12 protein localization is predominantly cytosolic and nuclear without demonstrable mitochondrial associa- tion, in human astrocytes and glioma cells. Other studies have shown that both BCL2L12 and BCL2L12-A isoforms are mainly local- ized to the nucleus of various human cell lines (HeLa, MCF-7, MDA- MB-231, and 293 T cells) (Hong et al., 2010), unlike other members of the BCL2 family, which predominantly localize to cytoplasm and Gene 505 (2012) 153–166 Abbreviations: BCL2L12, BCL2-like 12; BH2 domain, BCL2-homology-2 domain; BH3- like motif, BCL2-homology-3-like motif; HSPB5, heat shock protein beta-5; UV, ultraviolet; aa, amino acid(s); HSP70, heat shock 70-kDa protein; DFS, disease-free survival; OS, overall survival; NPC, nasopharyngeal carcinoma; CLL, chronic lymphocytic leukemia; EST(s), expressed sequence tag(s); cDNA, DNA complementary to RNA; CO 2 , carbon dioxide; FBS, fetal bovine serum; ku, kilo-units; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; oligo(dT), oligodeoxythymidine; DTT, dithiothreitol; dNTP, deoxyribonucleoside triphosphate; u, unit(s); RNase, ribonucle- ase; PCR, polymerase chain reaction; UTR, untranslated region; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EtdBr, ethidium bromide; ORF, open read- ing frame; PXXP motif, Pro-any-any-Pro motif; nt, nucleotide(s); 3D, three-dimensional; PTC, premature translation termination codon; NMD, nonsense-mediated mRNA decay; BH1 domain, BCL2-homology-1 domain; BH4 domain, BCL2-homology-4 domain; CDKN1A, cyclin-dependent kinase inhibitor 1A; ARE, AU-rich element. ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Panepistimiopolis, Athens 15701, Greece. Tel.: + 30 2107274306; fax: + 30 2107274158. E-mail address: ascorilas@biol.uoa.gr (A. Scorilas). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.04.084 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene