Figure 1

Splicing isoforms and expression pattern of NPPA and NPPA-AS. (A) Genomic structure of NPPA and its antisense transcript, NPPA-AS (represented by several overlapping ESTs). Arrowheads indicate the direction of transcription, vertical lines depict the overlap between sense and antisense exons. The length of complementary region is shown in basepairs. Coding regions of NPPA and putative NPPA-AS ORF are represented as black boxes, noncoding exons and UTRs as white boxes. (B) The expression analysis of NPPA demonstrates the presence of intron-retained variants (NPPA+Intr1+2, 1820 bp and NPPA+Intr1, 722 bp) in a number of tissues. PCR reactions were performed using the primers NPPA-F and NPPA-R (indicated by arrows labeled F and R on Figure 1C) and resolved on an agarose gel. Expected size of correctly spliced NPPA product is 600 bp. The shorter product in testis (457 bp) represents a testis-specific isoform of antisense transcript NPPA-AS.9, which was not represented by ESTs in the databank and therefore could not be avoided when designing primers. (C) The schematic depiction of NPPA and NPPA-AS splicing isoforms, the products of 3' RACE reactions and their location on the genomic sequence. The gray block arrows indicate the direction of transcription. (D) NPPA-AS is widely expressed as a complex combination of alternatively spliced isoforms. Upper panel: the agarose gel showing the reactions performed using primers NPPA-AS-F4 and NPPA-AS-R1 (indicated by arrows labeled F4 and R1 on Figure 1C) on the panel of tissue-specific cDNAs. Lower panel: Isoform 3'RACE.2 is expressed only in testis. PCR was performed using primers NPPA-AS-F1 and NPPA-AS-R5 (indicated by arrows labeled F1 and R5 on Figure 1C). The band corresponds to the expected size of 405 bp.