Coding region of adiponectin contains a silent intron reactivated by the adjacent intervening sequence of vector

Precise splicing pre-mRNA into correct mRNA is a tightly orchestrated process involving both cis and trans factors. However, the regulatory mechanism underlying alternative splicing remain elusive. An alternative splicing was revealed by comparing RT-PCR products (cDNA) of human adiponectin gene (ADPN) genes and sequencing the corresponding cDNA recovered from CHO-K1 cells transfected with a pIRES-neo vector carrying the cDNA. We determined that an 88-nt sequence in the original cDNA was missing from the adiponectin mRNA isolated from the transfected cells. After analyzing the flanking sequences and context of the 88-nt fragment, we discovered that it does have a typical intron configuration containing the splicing donor and acceptor, polypyrimidine tract, and branch site. A point mutation at the acceptor site (AG→TG) abolishes this splicing site indicating that it is a bona fide intron. The intron splicing defaulted again when the adjacent intervening sequence (IVS) of pIRES-neo was deleted or adiponectin 3'-UTR was present. We found that 3'-UTR segment contained several splicing silencers and IVS contained high density of splicing enhancers. It explained the reactivation of this silent intron. Our results elicited the possibility that a 3'-UTR-free cDNA may reactivate an otherwise silent intron in the coding region as it is cloned for expression in mammalian cells.