Novel Promoters for Human NAIP Figure 4. Expression of full-length NAIPJb transcripts across many tissues. At top, a schematic diagram of the 39 terminus of NAIP is shown, not to scale. Exons are indicated by black boxes, checkered and spotted arrows indicate the polarity of SINEs and LINEs, respectively. Not all repeat elements are shown. The arrowheads represent primers used to assess full-length NAIP transcription. Due to the high copy number of Alus in the human genome, the resultant RT-PCR gels were resolved by Southern blotting, with the unique probe shown, across the indicated tissues to reveal true AluJb-derived NAIP transcripts. doi:10.1371/journal.pone.0005761.g004 terminal BIR domain, followed by the central NBD, but only NAIP2 harbours C-terminal LRRs. Indeed, western blots on human PC3, HeLa, and NTera2D1 cell lysates indicate the presence of multiple bands corresponding to the above computer predictions (Figure 5b). To more accurately assess the potential for translation of the Alu-derived NAIP2 ORF we generated a NAIP:hemagglutinin fusion protein (HA:NAIPAlu) and over- expressed it in the cell lines indicated above. The recombinant protein HA:NAIPAlu is translated and migrates at 110 kDa with the putative endogenous isoform (NAIPAlu) in untransfected PC3 and HeLa cells (Figure 5b). It is clear the NAIP protein isoforms are differentially expressed in the queried cell lines, but all three cell lines endogenously produce the ,160 kDa NAIPfull and ,110 kDa NAIPAlu proteins, albeit to a different degree. In the PC3 and HeLa cell lines, where HA:NAIPAlu was overexpressed, an increase in band intensity is seen compared to NAIPAlu in untransfected cells. Overall, expression of the putative NAIPAlu protein is low relative to NAIPfull in all cell lines, however, the difference is not as exaggerated in NTera2D1 cells compared to PC3 or HeLa. Lastly, it appears that neither NTera2D1 nor HeLa cells express the putative ,130 kDa NAIP2 protein isoform. NAIP protein isoforms are broadly expressed in human tissues The observation that NAIP proteins equivalent in size to all of the computer-predicted isoforms are expressed in the cell lines screened, prompted a similar investigation of primary human tissues (Figure 6). A variety of NAIP proteins were detected in most of the tissues examined, although NAIPfull is not broadly expressed. In fact, NAIPfull was only detected in heart, skeletal muscle, and at very low levels in testis. Similarly, the ,110 kDa protein, which is expected to represent the Alu-derived NAIP ORF, is also only detected in heart and skeletal muscle. Potential NAIP2 proteins at ,130 kDa are observed almost uniformly across the tissues tested, and could correspond to NAIPGUSBP1-initiated transcripts. The subtle variation of the putative NAIP2 proteins, such as in spleen and heart, could result either from alternative start codon selection (Figure S6) or alternative splicing of NAIP2 terminal exons. Importantly, all of the tissues screened here, other than testis, derive from one individual with unknown NAIP copy number and mRNA expression levels. Nonetheless, we demon- strate the expression of various human NAIP protein isoforms that correspond with calculated molecular weights of the ORFs generated by alternative promoter usage. Discussion Transposable elements were initially discovered as important factors in the regulation of gene expression in maize, and termed controlling units [44]. This view of TE usefulness was contrasted by the ‘junk DNA’ hypothesis [45]. In recent times their practicality has garnered increased attention, particularly as mobile regulatory modules [5,9,13,30]. Strikingly, TEs are associated with many evolutionarily constrained regions in mammalian genomes [46], and many conserved non-coding elements are reported to function as transcriptional enhancers [47]. In general, it is difficult to ascertain the extent to which TEs donate their embedded regulatory signals to cellular genes, particularly because they can impose their effects over great distances. However, bioinformatics analyses of human and mouse genomes indicate a substantial impact of TEs on cellular gene regulation; as many as 25% of genes possess TEs in their UTRs [8,48]. Therefore, their influence on increasing the diversity of mammalian transcriptomes is likely underappreciated. The LTRs and LINEs, due to the natural presence of RNA pol II signals, are likely candidates to fulfill a regulatory role for cellular genes; dozens of known cases confirm their utility as regulatory modules [5,7,8]. In contrast, the pol III-dependent SINEs are concentrated in gene dense regions [1,6], but have largely been neglected as modulators of cellular gene expression. Recent bioinformatics analyses, however, have revealed the presence of numerous RNA pol II transcription factor binding sites and hormone response elements within SINEs [13,14], substantiating an earlier report [49]. Notably, the primate-specific Alus – divided into the old AluJ, intermediate AluS, and young AluY subfamilies – present consensus transcription factor binding sites distributed in an age-dependent manner [13]. Interestingly, among all gene-associated Alus on chromosome 21 and 22, older elements tend to harbour estrogen response elements and AP-1 docking sites, while younger and/or polymorphic Alus are PLoS ONE | www.plosone.org 6 June 2009 | Volume 4 | Issue 6 | e5761