Novel ADAM22 transcript variants isolated from the peripheral nervous system
In the case of human ADAM22, isolation of five splicing variants and existence of two terminating exons have been reported [13,20,21]. However, the latter terminating exon (exon 31) of the mouse species has not been identified yet. To isolate the long form of mouse ADAM22 transcripts, at first, we sought the mouse genome by BLAST homology search. Search results showed that the mouse genome contig NT_039297 contained most of the mouse Adam22 gene. In this contig sequence, we found putative exons 30 and 31, those sequences were quite similar to human ADAM22 isoform 1 (GenBank # NM_021723). To confirm existence of transcripts, we designed primers on the putative exon 31 and performed RT-PCR using mouse cerebellum mRNA as a template. TA-cloning of the amplified fragments and sequencing analysis showed that 5 of 8 clones were type 1 isoform (G01), 2 clones were type 3 isoform (G03) and 1 clone (G08) contained a novel exon between exons 29 and 30. Next, we performed RT-PCR analysis of mRNAs purified from several adult mouse tissues, such as the spinal cord, dorsal root ganglion (DRG), sciatic nerve and cultured primary Schwann cells. Interestingly, multiple DNA fragments were amplified from each tissue (Fig. 8), and length of major fragments in each lane was different. For example, length of major amplified PCR fragments from the sciatic nerve (Fig. 8A, lane 5) and cultured Schwann cells (lane 6) were almost identical, and were shorter than those from other tissues. In contrast, major fragments from the DRG (lane 4) were longer than others. We performed TA-cloning and sequencing to analyse exon organization of the amplified transcripts (Fig. 8C). Forty seven clones were isolated in total and were classified in 16 independent clones (G01 – G23). Comparison with the genomic sequence revealed the existence of 8 novel exons (exons 27S, 27L, 29.1, 29.3, 29.5, 29.7, 30 and 31). These novel exons were flanked by well-defined introns that obey the GT-AG rule. Exon organization of each clone was shown in Fig. 8C. Characteristic feature of ADAM22 transcripts is tissue specific insertion or skipping of exons. Because the number of nucleotides of exons 26, 27, 27L, 29.3, 29.5 and 29.7 is a multiple of 3, insertion or skipping of these exons will not change the downstream reading frame. Known peptide motives were not found in the newly isolated sequences. This RT-PCR analysis is summarized as follows: ADAM22 transcripts were roughly subdivided in 3 groups, CNS-form (containing exon 27), DRG-form (containing exon 27L) and Schwann-cell-form (skipping exon 26 and 27). From these results, we concluded that ADAM22 is expressed in a cell-type specific manner, and plays an essential role in myelinogenesis in the PNS.