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Identical transcription initiation sites for proinsulin messenger ribonucleic acid in three insulin-expressing
Abstract:
Proinsulin mRNA was analyzed by RNA blot hybridization from four insulin-expressing tissues of the rat, including adult and fetal pancreas, yolk sac, and an insulinoma cell line (RIN 5F). The proinsulin mRNA transcripts from the insulinoma cell line and fetal pancreatic tissue were estimated to be, respectively, 100 and 50 bases larger than the transcript from adult pancreas. Yolk sac proinsulin mRNA comigrated with the fetal transcript. While glucose is an important regulator of proinsulin mRNA in the adult, there is a marked increase in the concentrations of proinsulin mRNA and insulin in the developing rat, although plasma glucose levels are quite low. Expression of proinsulin mRNA independent of glucose levels is also found in insulinoma tissue. In addition, there is a second TATA sequence upstream of the putative start site in rat insulin II gene, and the transcription initiation site(s) has not been mapped in all of these tissues. These observations suggested that alternative transcription initiation sites may place the genes under different promoter control during development. To map the 5' end of the gene, primer extension was performed using a synthetic oligonucleotide primer complementary to the first 20 bases of the coding portion of the rat insulin I gene. The extended products of the proinsulin mRNAs from adult insulinoma cell line and fetal pancreas were identical and consistent in size with those predicted if transcription occurs at the putative start site. The 3' ends of the proinsulin mRNA transcripts were evaluated by ribonuclease H digestion, and it was shown that the noted size differences could be accounted for by different lengths of 3'-polyadenylation. Northern blot analysis of proinsulin mRNA from animals treated under conditions where mRNA varied from low to high levels failed to show any modulation of polyadenylation. The role of polyadenylation of proinsulin mRNA in the physiological regulation of insulin biosynthesis, if any, is currently unknown.