title: Degradation of apolipoprotein II mRNA occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-noncoding region.
abstract: Degradation intermediates of the estrogen-regulated apolipoprotein (apo) II mRNA were identified by S1 nuclease mapping and primer extension analysis. S1 mapping of poly(A)-RNA detected a series of mRNAs truncated at specific sites in the 3'-noncoding region. Many of these sites were also detected by primer extension analysis indicating that truncated molecules resulted from endonucleolytic cleavage in the 3'-noncoding region. Identical cleavage sites were seen with RNA from estrogen-treated animals or from animals withdrawn from hormone under conditions where apoII mRNA degraded in the slow (t1/2 = 13 h) or rapid (t1/2 = 1.5 h) decay mode. No differences were seen in poly(A) tail length or heterogeneity among these conditions. These results indicate that the estrogen-induced alteration in apoII mRNA turnover does not involve a new pathway of degradation, but, more likely, involves an increased targeting of the mRNA for degradation by a preexisting pathway. These data are consistent with a mechanism in which the initial step in apoII mRNA degradation is an endonucleolytic cleavage in the 3'-noncoding region without prior removal of the poly(A) tail. The endonucleolytic cleavage sites occurred predominantly at 5'-AAU-3' or 5'-UAA-3' trinucleotides found in single-stranded domains in a secondary structure model of the naked mRNA (Hwang, S-P. L., Eisenberg, M., Binder, R., Shelness, G. S., and Williams, D. L. (1989) J. Biol. Chem. 264, 8410-8418). The structure of the 3'-noncoding region in polyribosomal messenger ribonucleoprotein was examined by titrations of liver homogenates with dimethyl sulfate and cobra venom RNase. The results suggest that the typical cleavage site is a 5'-AAU-3' or 5'-UAA-3' trinucleotide in an accessible single-stranded loop domain. Single-stranded domains alone or accessible domains alone are not sufficient for cleavage. Similarly, 5'-AAU-3' or 5'-UAA-3' trinucleotides alone are not sufficient for cleavage. Localization of these trinucleotides to accessible single-stranded domains in the polyribosomal messenger ribonucleoprotein may provide the specificity for cleavage during targeted degradation.
authors.affiliation: Department of Pharmacological Sciences, University of New York, Stony Brook 11794.
authors.forename: R
authors.initials: R
authors.lastname: Binder
authors.forename: S P
authors.initials: SP
authors.lastname: Hwang
authors.forename: R
authors.initials: R
authors.lastname: Ratnasabapathy
authors.forename: D L
authors.initials: DL
authors.lastname: Williams
mesh_terms: D000818:Animals; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A / Q000235:genetics*; D001483:Base Sequence; D002645:Chickens; D004720:Endonucleases; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009710:Nucleotide Mapping; D011132:Polyribosomes / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000648:ultrastructure; D015719:Single-Strand Specific DNA and RNA Endonucleases
chemical_list: D016633:Apolipoprotein A-II; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases