A Novel Protein Isoform of the Multicopy Human NAIP Gene Derives from Intragenic Alu SINE Promoters Mark T. Romanish1,2, Hisae Nakamura3, C. Benjamin Lai1,2, Yuzhuo Wang3,4,5, Dixie L. Mager1,2* 1 Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada, 2 Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, 3 Cancer Endocrinology, BC Cancer Agency, Vancouver, British Columbia, Canada, 4 Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada, 5 The Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia, Canada Abstract The human neuronal apoptosis inhibitory protein (NAIP) gene is no longer principally considered a member of the Inhibitor of Apoptosis Protein (IAP) family, as its domain structure and functions in innate immunity also warrant inclusion in the Nod- Like Receptor (NLR) superfamily. NAIP is located in a region of copy number variation, with one full length and four partly deleted copies in the reference human genome. We demonstrate that several of the NAIP paralogues are expressed, and that novel transcripts arise from both internal and upstream transcription start sites. Remarkably, two internal start sites initiate within Alu short interspersed element (SINE) retrotransposons, and a third novel transcription start site exists within the final intron of the GUSBP1 gene, upstream of only two NAIP copies. One Alu functions alone as a promoter in transient assays, while the other likely combines with upstream L1 sequences to form a composite promoter. The novel transcripts encode shortened open reading frames and we show that corresponding proteins are translated in a number of cell lines and primary tissues, in some cases above the level of full length NAIP. Interestingly, some NAIP isoforms lack their caspase- sequestering motifs, suggesting that they have novel functions. Moreover, given that human and mouse NAIP have previously been shown to employ endogenous retroviral long terminal repeats as promoters, exaptation of Alu repeats as additional promoters provides a fascinating illustration of regulatory innovations adopted by a single gene. Citation: Romanish MT, Nakamura H, Lai CB, Wang Y, Mager DL (2009) A Novel Protein Isoform of the Multicopy Human NAIP Gene Derives from Intragenic Alu SINE Promoters. PLoS ONE 4(6): e5761. doi:10.1371/journal.pone.0005761 Editor: Mark A. Batzer, Louisiana State University, United States of America Received April 6, 2009; Accepted May 6, 2009; Published June 2, 2009 Copyright: 2009 Romanish et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grant #10825 to DM and grant #86730 to YW from the Canadian Institutes of Health Research (http://www.cihr.ca), with core support provided by the BC Cancer Agency. MR is supported by a studentship from the Michael Smith Foundation for Health Research (http://www.msfhr. org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dmager@bccrc.ca Introduction Transposable elements (TEs) are ubiquitous components of most sequenced genomes, but their function, if any, is poorly understood. Comprising ,50% of the human genome, the majority of TEs belong to the short interspersed element (SINE) (.10%), long interspersed element (LINE) (.20%), and endog- enous retroviral/long terminal repeat (LTR) (,10%) families [1]. The SINEs encode no open reading frame (ORF) and have utilized LINE-encoded proteins [2] to amplify to .106 copies in the human and mouse genomes [1,3]. On the other hand, only a limited number of LINEs and LTR elements are full-length; many of which are rendered non-functional due to point mutations and deletions [4]. Therefore, the majority of TEs no longer pose a significant burden as insertional mutagens, although many retain the regulatory signals necessary for transcription [5,6]. The LTRs and LINEs naturally harbour RNA polymerase II (pol II) signals and numerous examples of promoter exaptation by host genes exist [5,7,8]. On the other hand, SINEs replicate via pol III [9], and thus are not expected to impose direct regulatory effects on protein-coding genes. Indeed, SINEs are over- represented within gene-rich regions, while the LTRs and LINEs are under-represented [6]. Recent scrutiny of the primate-specific Alu SINEs has provided various illuminating findings. They can be incorporated into mRNA as cassette exons [10,11], and are often found in UTRs [8,9,12]. Furthermore, consensus binding motifs for many pol II transcription factors have recently been identified within Alus [13,14], but their role as promoters and enhancers has not been extensively researched. We have previously shown that the neuronal apoptosis inhibitory protein (NAIP) orthologues in human (NM 022892.1) and mouse (NM 008670.2; NM 021545.1; NM 010870.2; NM 010872.2) provide a remarkable example of LTR promoter exaptation – unrelated LTRs were independently acquired as gene promoters [15]. NAIP is a member of the inhibitor of apoptosis protein (IAP) family, and was cloned as a candidate gene for the neurodegenerative disorder Spinal Muscular Atrophy (SMA) [16]. Consistent with its role as a modifier of SMA severity, NAIP has been shown to inhibit programmed cell death by binding activated caspases [17,18,19]. Moreover, the IAPs have emerged as therapeutic and diagnostic targets for various cancers [20,21,22]. Furthermore, the effect of NAIP expression in other neurodegen- erative diseases, such as Alzheimer’s disease, Down syndrome, multiple sclerosis, and Parkinson’s disease, has also been investigated [23,24]. Recently, a potential role in innate immunity surfaced through the discovery that polymorphism of a particular Naip copy in mouse strains determined permissiveness of Legionella pneumophila replication in host macrophages [25]. Paradoxically, Naip-mediated L. pneumophila restriction is caspase 1-dependent and signaling through this pathway results in the rapid death of PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5761