Novel Promoters for Human NAIP neighboring TSS, map with perfect identity to NAIPfull, NAIP1, and NAIP2. B) Novel regulatory regions associated with NAIP transcription. Luciferase assays were performed using reporter constructs centered on the previously identified ERV-P and NAIPfull, and the NAIPSg and NAIPJb TSS identified here (indicated by bent arrows). The fragments tested are denoted by solid bars beneath the magnified NAIPfull image (top), and are labeled accordingly. Exons, Alus, and LTR elements are indicated as in Figure 2a; here, LINE fragments are indicated as speckled arrows. Values are normalized to an internal control (Renilla luciferase) and expressed relative to a promoter-less control vector (pGL3-Basic). Each bar represents the mean of at least four independent experiments 6 SD. Gene diagrams are not drawn to scale. doi:10.1371/journal.pone.0005761.g002 from the same patient. Perhaps transcription in liver produces isoforms that constitutively omit one or both exons to which our 39 qRT-PCR primer sets are designed. Alternatively, NAIPfull transcripts in this tissue could be aberrantly poly-adenylated. Regardless, neither NAIPSg nor NAIPJb are highly expressed in liver. Full-length Alu-derived transcripts are broadly expressed The fact that the AluJb functions as a pol II promoter is an intriguing finding, with genome-wide ramifications in establish- ment of transcriptional networks, as previously suggested [13,30]. We next examined the potential for transcription of a novel NAIP ORF as a result of Alu promoter activity. Indeed, if all downstream exons are included in at least some Alu-derived NAIP transcripts, a 2,643 nucleotide ORF is preserved (Figure S6). Therefore, we sought to determine whether Alu-initiated transcripts continue to the 39 terminus, by RT-PCR. Southern blotting was required since, by necessity, primers hybridized to Alus – the most plentiful elements in primate genomes [1]. Across all tissues screened, except liver, products corresponding to the expected size (,3 kb) were resolved for NAIPJb (Figure 4). Among various minor forms, one notable variant of ,2 kb is expressed at the same frequency as full-length NAIPJb. This ,2 kb variant, among numerous others including full-length, is also observed for NAIPSg transcripts in several tissues (data not shown). Potentially the smaller isoform could result from alternative splicing common to both NAIPJb and NAIPSg transcripts, between the site of reverse primer binding and probe hybridization. Alternatively, a single NAIP transcript possessing a second exonized Alu downstream of some or all of the probe-binding region could also explain this observation. The prominent ,3 and ,2 kb bands do not result from the simultaneous amplification of NAIPJb and NAIPSg due to primer cross-reactivity, since the respective transcripts and their unique 59 UTRs are roughly equal in size. Nonetheless, existence of full- length Alu-derived transcripts, a potential 2,643 nucleotide ORF, and numerous in-frame ATGs in accordance with derived consensus sequences [42,43] (Figure S6) suggest a potential for the synthesis of NAIP protein isoforms. Novel human NAIP protein isoforms Using the annotated copies of NAIP in the sequenced human genome as a reference [34], we scanned all possible full-length transcripts that could arise from the novel TSS reported above for ORFs and domain composition. Many potential ORFs were identified for each queried transcript, but only the longest examples were considered. Interestingly, all accepted examples represented N-terminal truncations of NAIPfull, indicating the existence of numerous potentially functional in-frame translation initiation codons (Figure 5a, Figure S6). NAIPfull was previously shown to comprise 1403 amino acids and yield a ,160 kDa protein encoding three N-terminal anti-apoptotic Baculoviral IAP Repeat (BIR) domains, followed by a central nucleotide binding domain (NBD) and C-terminal leucine-rich repeats (LRR) [16]. NAIPSg- and NAIPJb-mediated transcription of NAIP2 is predicted to generate an ORF 881 amino acid long, and corresponds to a 110 kDa protein that excludes the BIRs (NAIPAlu). Due to the deletion of exons 12-14 in NAIP1 a C-terminal truncation of the LRRs is also predicted, in addition to a truncation of its N terminus (Figure 1b), and could produce a ,85 kDa NAIP protein isoform, but was not detected. Finally, transcription from the promoter within the final GUSBP1 intron can drive expression of both NAIP1 and NAIP2, and potentially gives rise to 100 kDa (NAIP1) and 130 kDa (NAIP2) proteins, respectively. Both putative protein isoforms, NAIP1 and NAIP2, possess one N- Figure 3. Contribution of Alu-initiated isoforms to total NAIP transcription. Expression levels of the targets: NAIPTotal (39), NAIPfull (59), NAIPJb, and NAIPSg were normalized to b-actin and are shown relative to 39 levels of NAIP transcription in the indicated tissues. Each bar represents the mean of at least five independent experiments 6 SD. doi:10.1371/journal.pone.0005761.g003 PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e5761