Expression of NAIP in these tissues most likely results from macrophage infiltration [35], the cell type mediating NAIP- dependent L. pneumophila immunity. The largest difference is observed in testis where 39 levels are .40-fold above 59 levels. Interestingly, in liver 59 levels of NAIP are the highest (Figure 1c), potentially arising from transcription of 39 deleted isoforms, premature poly-adenylation, or CNV-associated anomaly within the tissue sample screened. The abundance of 39 transcripts raises the possibility that the 59 deleted copies, NAIP1 and NAIP2, are expressed (Figure 1c, Figure S2), or that internal promoters of NAIPfull produce transcripts lacking the 59 end, or both. Novel human NAIP transcription start sites The observation that levels of 59 vs. 39 transcription are not uniform across various human tissues prompted an analysis to determine where NAIP transcription was initiating. Previously, we showed that an upstream ERV-P LTR is a promoter of NAIPfull specifically in testis, but that ubiquitous expression derives from within an exon in the 59 UTR [15]. Moreover, a previously published transcription start site [36], overlaps a MER21C LTR slightly upstream of the ERV-P, but could not be confirmed by 59 RACE. However, an RT-PCR approach using tiled primers, similar to that of Xu et al. [36], indicated that an adjacent AluSx SINE was also included in these transcripts (Figure S3). We are unable to conclude whether this SINE is in fact a site of NAIP transcription or an internal exon of an undescribed 59 UTR. Here we revised our previous 59 RACE approach, which only assessed the transcription start sites (TSS) associated with expression of NAIPfull [15], and numerous novel TSS were discovered (Figure 2). Unexpectedly, we observed that two Alu SINEs localized 59 of exon 10, an AluSg and AluJb, are sites of NAIP transcriptional initiation, hereon referred to as NAIPSg and NAIPJb (Figure 2a). These Alus are in the antisense orientation, full- length (,300 bp) and present in NAIP orthologues of New and Old World primates (data not shown). Since sequence identity hinders their unambiguous mapping, NAIPSg and NAIPJb 59 RACE clones could arise from three of the five copies (NAIPfull, NAIP1, and NAIP2) in the reference human genome (Figure S4). Thus, either NAIP1 and/or NAIP2 are expressed from Alus, or these Alus may serve as promoters within NAIPfull, or both. A number of NAIPSg clones were obtained that mapped to two distinct TSS localizing in the 39 terminus of the Alu (Figure S4a). Interestingly, the AluSg A-rich tail is known to be hypermutable [37,38], however, the corresponding region of this particular element is identical to its consensus sequence. The upstream ,9 kb (relative to NAIPSg polarity) is a patchwork of LINE fragments and Alus, and likely contributes additional regulatory signals. All NAIPSg clones splice into the adjacent exon 8 (Figure 2a, Figure S4a), utilizing a splice donor site frequently employed by exonized antisense Alus [10,11]. Several NAIPJb clones were also obtained, these map to two particular regions localized near the AluJb 59 terminus (Figure S4b). The regulatory signals comprising the NAIPJb core promoter, therefore, are expected to lie within the body of this Alu. The NAIPJb clones, however, do not splice into the downstream exon 10, rather transcription continues through the intervening ‘intron’. The validity of NAIPJb transcripts is verified by +/2 RT controls (Figure S5). Interestingly, the splice donor sequence utilized by NAIPSg has undergone an AGRAT transversion mutation in NAIPJb (Figure S4b); its capacity for splicing has not been studied here. Additional TSS downstream of NAIPJb, in the intervening sequence adjacent exon 10, are also observed (Figure S4b). Another site of transcription initiation was identified within the final intron of the GUSBP1 gene (Figure 2a). Although sequence Novel Promoters for Human NAIP identity hinders unambiguous mapping of this transcript, the novel first exon splices into exon 4 of the adjacent NAIP1 and/or NAIP2. Consequently, expression of at least one other NAIP copy, in addition to NAIPfull, is demonstrated since a TSS within the final intron of the GUSBP1 gene is only adjacent to NAIP1 and NAIP2. Promoter activity of proximal NAIPSg and NAIPJb sequences Particularly intrigued by the Alu TSS, we tested the capacity of the underlying sequences as pol II promoters in reporter gene assays, relative to the 59 promoters we previously identified [15]. Indeed, the ubiquitous NAIPfull and LTR-derived, testis-specific NAIPERV-P are capable promoters in the NTera2D1, HeLa (Figure 2b), and Jeg3 (data not shown) cell lines. A .500 bp DNA fragment underlying the NAIPJb TSS, including the ,200 bp of upstream Alu sequence and extending 59 toward exon 10, exhibits strong promoter activity (Figure 2b). Similarly, a 600 bp fragment centered on the NAIPSg TSS, containing the entire AluSg and the upstream 300 bp of internal L1 sequence, also exhibits considerable promoter activity relative to an empty vector control, in fact comparable to the LTR (Figure 2b). Due to location of the AluSg TSS, the upstream L1 fragment likely contributes promoter regulatory motifs, but its position relative to a full-length L1 does not correspond to the previously described antisense L1 promoter [7]. Analysis of the nucleotide sequences underlying the NAIPSg and NAIPJb TSS revealed the incidence of several putative pol II regulatory motifs, including: TATA-like boxes, initiator sequences, and downstream promoter elements (Figure S4) [39]. Accumulating evidence indicates that numerous pol II transcription factor binding sites lie within Alu elements [13,14]. Indeed, both NAIP-associated Alus possess potential AP-1 and retinoic acid- and estrogen response element binding motifs (Figure S4a,b), in agreement with published consensus sequences [13]. Variable contribution of Alu-associated NAIP transcripts in different tissues To address the contribution of Alu-derived NAIP transcripts to total NAIP expression, qRT-PCR was performed. Although their transcription is detected in most tissues screened by RT-PCR (Figure S5), this approach indicates NAIPJb is expressed at levels similar to or higher compared to NAIPfull in many of the tissues tested, and is therefore likely an important promoter (Figure 3). In contrast, NAIPSg does not contribute significantly to total NAIP expression in any tissue tested (Figure 3). Interestingly, scrutiny of 59 RACE sequences revealed that NAIPSg undergoes RNA editing in its 59 UTR (Figure S4a), a common observation among transcribed Alus [40,41]. Comparison of edited vs. un-edited NAIPSg transcript levels indicated the former is .10-fold more abundant than the latter (data not shown). Most NAIP transcription in colon, spleen, lung, and prostate could be accounted for by the combined activity of all queried promoters, but the contribution of individual paralogues could not be assessed due to their high sequence identity. However, in kidney and testis all isoforms are not detected and it is likely that unaccounted 39 transcription either initiates downstream of AluJb, as indicated above (Figure S4b), or from the NAIPGUSBP1 TSS. Contribution of NAIPGUSBP1-derived transcripts could not be assessed due to the complexity of alternative splicing in this 59 UTR (Figure S5). As discussed previously, the 59 levels of NAIP in liver are expressed 4-fold over 39 levels, suggesting that all transcription in this tissue derives from NAIPfull. Since two independent liver RNA samples were screened, this rules out the possibility of patient-specific CNV, unless both samples derive PLoS ONE | www.plosone.org 3 June 2009 | Volume 4 | Issue 6 | e5761