# Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (_SLC34A1_/Pfn3)
## 3 Discussion
Our observations that the _SLC34A1_-encoded phosphate transporter was difficult to detect in phosphate uptake experiments indicate that also in our HEK293 cell model, the _SLC34A1_ mRNA is not efficiently translated. The levels of _PFN3_ mRNA are even considerably low in HEK293 cells and localized in the cell nucleus; nevertheless, we cannot rule out that the _PFN3_ gene is protein coding. In fact, immunological detection of PFN3 protein has been reported in mouse testes in the final stages of sperm development [40]. Moreover, disruption of _Pfn3_ causes abnormal acrosome formation and leads to subfertile male mice [41]. Antisense transcripts complementary to _SLC34A_ loci have been studied in various model systems, including zebrafish embryos, _Xenopus_ oocytes, mice and human cell lines [7; 22; 23; 42]. Initial expression analyses focused on testes and kidneys where the antisense and sense transcripts, respectively, are predominantly detected [43]. However, evidence that an antisense-mediated mechanism contributes to the physiological regulation of the sense-encoded phosphate transporter and phosphate homeostasis proved inconclusive. Accordingly, _SLC34A_ sense and antisense transcripts show different expression patterns; the sense transcripts are present in epithelial tissues of the kidney (_SLC34A1_) and intestine (_SLC34A2_, mammals and fish), and the antisense RNA is most prominently expressed in tests [44; 45]. Co-expression has been detected during zebrafish development and also in mouse testis and kidney, though the biological rationale for the expression may not be the related to Pi homeostasis (see below) [22]. During a window of co-expression in zebrafish, endo-siRNA's were detected, pointing to an antisense mediated regulatory mechanism that involves dsRNA formation and processing by Dicer [46]. However, this mechanism appears to not apply to the cell culture model described here, since the gene pairs producing endo-siRNA's are usually antagonistically regulated and we were unable to detect _SLC34A1_/_PNF3_-derived small RNAs on Northern blots (not shown). In the early days of the genomic era, NATs were considered a separate family of non-protein coding transcripts, distinct from other long noncoding RNAs with a specific biological rationale and potentially common mechanistic themes. The mechanisms discussed, i.e., the various modes of transcriptional interference and post transcriptional sense-antisense interactions, have been briefly outlined in the introduction and are extensively reviewed [3; 11; 13]. An increasing number of recent studies have focused on mechanistic aspects of sense and antisense expression, especially in the context of cancer. Two types of strategies clearly prevailed: The function as a microRNA sponge and an interaction of the antisense transcript with chromatin-modifying enzymes. The importance of the former mechanism has only emerged in the past few years, predominantly in the context of cancer. A common experimental strategy identifies a differentially expressed antisense transcript in cancer vs. untransformed cells followed by miRNA target gene prediction and experimental validation [14], ideally demonstrating the direct interaction between miRNA and target antisense transcript [47]. Alternatively, immunoprecipitation of Ago2 followed by RT-qPCR was used to corroborate the interaction between the miRNA and its target antisense transcript [48]. The regulatory mechanism where antisense transcripts induce chromatin modifications prominently involves polycomb repressive complex 2 (PRC2). PRC2 induces H3K27 trimethylation [49], which could relate to the decrease in H3K27 acetylation that we observed at the antisense promoter in response to dexamethasone (Figure 4) [50]. Molecular aspects of PRC2-mediated gene silencing are well established in the context of X chromo some inactivation and the noncoding RNAs _Xist_ and _Tsix_, the antisense transcripts _Ain_ and Kcq1 ot1 in parental imprinting, as well as the antisense lncRNAs _Hotair_ and _Anril_[51; 52; 53].