enriched for other features, including retinoic acid response elements. In addition, important roles in mRNA poly-adenylation have also been revealed for Alus and other TEs in a variety of organisms [50,51]. Since Alus number .106 copies in the human genome, are enriched in gene-dense regions, and contain potential pol II transcriptional regulatory motifs, they could be considered the most important transcriptional regulators. For the first time it is shown here that an Alu can function as a direct promoter for a human gene. More commonly, they and other SINEs are incorporated into mRNA UTRs and coding regions as cassette exons [5,8,9,52], facilitated by the presence of numerous splice donor and acceptor sites in the sense and antisense orientations [10]. Examples of SINE exaptation as promoters, however, are limited and represented by a sense B1 [53] and an antisense B2 [54] element in mouse. In human, an isoform of the p75TNFR gene initiates transcription from an antisense MIR SINE, with the adjacent AluJo providing an alternative translation start site [55]. Furthermore, a bioinfor- matics analysis reports the existence of several unvalidated antisense Alu-associated TSS [8]. Here, broad transcription of NAIP isoforms from exapted antisense AluJb and AluSg elements is demonstrated in a number of tissues, but it is unknown whether these sequences would also be functional in the sense orientation. The Sg and Jb exaptations associated with NAIP transcription belong to older families that exhibit 10% and 15% divergence from their consensus sequences, respectively. Remarkably, NAIPJb- associated transcripts are more highly expressed than full-length isoforms in many tissues, but NAIPSg levels are at the limit of detection. We further demonstrate that the Alu-initiated NAIP transcripts extend to the 39 terminus, and that the associated ORF, harbouring only NBD and LRRs, is translated in a variety of cell lines and primary human tissues. Our findings also suggest that the other predicted novel NAIP proteins are expressed, in addition to the BIR-less isoform directly assessed here. It is notable that the tissue blot we screened derives from one adult individual, with the exception of testis, indicated by the manufacturer as an accidental fatality. An earlier analysis of pooled primary human tissue samples using a different antibody, also revealed similar NAIP protein isoforms that were speculated to arise by alternative splicing [35]. Nonetheless, the data presented here substantiate transcriptome analyses that reveal alternative promoter usage as an important source of alternative mRNAs and proteins [56,57]. The NAIP gene first rose to prominence when it was cloned as a putative disease allele for the neurodegenerative disorder, Spinal Muscular Atrophy (SMA) [16], but is now understood to influence SMA severity, which is induced by the adjacent SMN gene [58]. Its identification did seed discovery of the Inhibitor of Apoptosis Protein (IAP) family in animals [19]. The IAPs sequester activated caspases, the agents of cell death, via their signature N-terminal BIR domains [20]. Interest in NAIP was renewed through the discovery that polymorphism of the murine Naip5 (Birc1e) copy solely determines permissiveness of Legionella pneumophila replication in host macrophages [25]. Human Legionella infections result in Legionnaire’s disease, a severe type of pneumonia [59]. It was recently shown that human NAIP also blocks L. pneumophila replication in cell lines and primary cells, suggesting a common function [60]. NAIP-dependent sensing of cytosolic microbial patterns is LRR-dependent, and is currently known to respond to Legionella and Salmonella typhimurium flagellin [26]. These and other findings point to an important role in the innate immune response, and justify the inclusion of NAIP in the NLR superfamily [29]. Invariably, the NLRs possess a central NBD and C-terminal LRRs; collectively they survey the cytosol for pathogen associated molecular patterns and elicit the appropriate response [61]. Novel Promoters for Human NAIP While the potential functions of the novel NAIP protein isoforms are unknown, there are several possibilities. Firstly, NAIP proteins are known to homo-oligomerize via their NBD [17], therefore, expression of BIR-truncated isoforms and their subsequent interaction with NAIPfull, could be a mechanism whereby its anti-apoptotic properties are effectively dispersed among a greater number of cytosolic molecules. Alternatively, these could be dominant negatives and serve to regulate the amount of anti-apoptotic NAIP molecules active in a given cell. Finally, expression of NAIP protein isoforms could represent a new example of innovation within the innate immune system, whereby hetero-oligomerization of NLRs creates diversity among these cytosolic sensors, analogous to the Natural Killer inhibitory cell receptor repertoire [62]. Indeed, NBD-mediated heterotypic interactions of some NLRs, including NAIP, have been demon- strated [63]. Moreover, Naip was also shown to co-precipitate with its closest homologue, ICE protease activating factor (Ipaf) [27]. Together these proteins activate Interleukin converting enzyme (ICE or caspase 1), and initiate caspase 1-dependent cell death in response to cytosolic flagellin [26,27,28]. Although caspase 1 is required to cleave the inflammatory cytokines proIL-1b and proIL-18 into their active forms, their involvement in this process remains unresolved. Interestingly, and perhaps not coincidentally, the cellular processes affected by IL 1b – proliferation, differentiation, and apoptosis – are the same as those influenced by AP-1 transcriptional regulation [64]. Genes involved in immunity tend to permit regulatory variation [8], as do multicopy genes [52]. While it is known that alternative 59/39 ends create genetic variation that leads to proteome evolution [56,57,65], the effect of Alu elements is under appreciated. Here we show that transcription from Alus generates a novel NAIP ORF that is subsequently translated, clearly indicating the effect they have on not only gene regulation, and perhaps establishment of transcriptional networks [13,30], but also proteome evolution. Methods Ethics Statement The blood sample was obtained with written informed consent according to a protocol approved by the University of British Columbia Research Ethics Board. RNA and Reverse Transcription With the exception of blood, all human RNA was purchased from Clontech (Mountain View); each sample consists of pooled material from multiple individuals. Blood was obtained from a healthy human adult with informed consent and the sample subsequently underwent erythrocyte reduction. RNA from remaining peripheral blood leukocytes (PBLs) was isolated using the QIAmp RNA Blood Mini Kit (Qiagen). Where necessary, RNA was isolated from candidate cell lines using TRIzol (Invitrogen) according to the manufacturer’s recommendations. Prior to reverse transcription, RNA was quantified using a Qubit fluorometer (Invitrogen). All cDNA synthesis was prepared by random hexamer-primed Superscript III Reverse Transcriptase (Invitrogen), as directed by the manufacturer. RT-PCR All RT-PCR, except as indicated below for amplification of the NAIP ORF and generation of the expression vector, was performed with Platinum Taq DNA Polymerase (Invitrogen) and the relevant primers are listed in Table S1, all used at 10 mM. Optimal primer annealing temperatures were deduced using the PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e5761