Role of the 5'-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine.

Abstract

S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5'-untranslated region (5'UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5'UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5'UTR, as well as chimaeric constructs in which the AdoMetDC 5'UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5'UTR, the AdoMetDC protein-coding region and the 3'UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5'UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5'UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5' end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5'UTR that may include but is not entirely due to the internal ORF.