An endonuclease activity similar to Xenopus PMR1 catalyzes the degradation of normal and nonsense-containing human β-globin mRNA in erythroid cells

Abstract

β-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 β-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 β-globin mRNA with a plasmid vector expressing the catalytically active form of PMR1 reduced the half-life of β-globin mRNA from 12 to 1–2 h without altering GAPDH mRNA decay. The reduced stability of β-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 β-globin mRNA. Therefore, β-globin mRNA is cleaved in vivo by an endonuclease with properties similar to PMR1. Inhibiting translation with cycloheximide stabilized nonsense-containing β-globin mRNA, resulting in a fivefold increase in its steady-state level. Taken together, our results indicate that the surveillance of nonsense-containing β-globin mRNA in erythroid cells is a cytoplasmic process that functions on translating mRNA, and endonucleolytic cleavage constitutes one step in the process of β-globin mRNA decay.