title: Coding region of adiponectin contains a silent intron reactivated by the adjacent intervening sequence of vector.
abstract: 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.
authors.affiliation: Department of Physiology, National Yang-Ming University, Taipei 11221, Taiwan, ROC.
authors.forename: Tzu-Ying
authors.initials: TY
authors.lastname: Liu
authors.forename: Chih-Ying
authors.initials: CY
authors.lastname: Chen
authors.forename: Chi-Chang
authors.initials: CC
authors.lastname: Juan
authors.forename: Lo-Chun
authors.initials: LC
authors.lastname: Au
mesh_terms: D020413:3' Untranslated Regions; D052242:Adiponectin / Q000235:genetics* / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D017354:Point Mutation; D022821:RNA Splice Sites; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; C499918:ADIPOQ protein, human; D052242:Adiponectin; D018076:DNA, Complementary; D022821:RNA Splice Sites