Dual function of the intron of the rat insulin I gene in regulation of gene expression.
Since the short intron in the 5'-untranslated region (5'-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5'-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5'-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins11VS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides -87 and +110 showed a reduction of expression by 30%. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.
Damert
A
A
Institute of Biochemistry, School of Medicine, E,-M.-Arndt University, Greifswald, Germany.
Leibiger
B
B
Leibiger
I B
IB
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D006224:Cricetinae; D004252:DNA Mutational Analysis; D017931:DNA Primers; D005786:Gene Expression Regulation; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics*; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D014162:Transfection
D017931:DNA Primers; D007328:Insulin; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase