An endonuclease activity similar to Xenopus PMR1 catalyzes the degradation of normal and nonsense-containing human beta-globin mRNA in erythroid cells.
beta-globin mRNA bearing a nonsense codon is degraded in the cytoplasm of erythroid cells by endonuclease cleavage, preferentially at UG dinucleotides. An endonuclease activity in polysomes of MEL cells cleaved beta-globin and albumin mRNA in vitro at many of the same sites as PMR1, an mRNA endonuclease purified from Xenopus liver. Stable transfection of MEL cells expressing normal human beta-globin mRNA with a plasmid vector expressing the catalytically active form of PMR1 reduced the half-life of beta-globin mRNA from 12 to 1-2 h without altering GAPDH mRNA decay. The reduced stability of beta-globin mRNA in these cells was accompanied by an increase in the production of mRNA decay products corresponding to those seen in the degradation of nonsense-containing beta-globin mRNA. Therefore, beta-globin mRNA is cleaved in vivo by an endonuclease with properties similar to PMR1. Inhibiting translation with cycloheximide stabilized nonsense-containing beta-globin mRNA, resulting in a fivefold increase in its steady-state level. Taken together, our results indicate that the surveillance of nonsense-containing beta-globin mRNA in erythroid cells is a cytoplasmic process that functions on translating mRNA, and endonucleolytic cleavage constitutes one step in the process of beta-globin mRNA decay.
Bremer
Kirsten A
KA
Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.
Stevens
Audrey
A
Schoenberg
Daniel R
DR
D000818:Animals; D018389:Codon, Nonsense; D003513:Cycloheximide / Q000494:pharmacology; D004722:Endoribonucleases / Q000378:metabolism*; D015672:Erythroid Precursor Cells / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D006801:Humans; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D014981:Xenopus
D018389:Codon, Nonsense; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D005914:Globins; D003513:Cycloheximide; D004722:Endoribonucleases; C116181:polysomal RNase 1