Quantitative analysis of deadenylation-independent mRNA decay by a modified MBRACE assay

Abstract

Endonuclease cleavage is the rate-limiting step in the decay of nonsense-containing human β-globin mRNA in erythroid cells. The 5′-truncated intermediates thus generated are polyadenylated and more stable than the parent mRNA. Northern blotting is commonly used to measure the decay rate of full-length mRNA, and S1 nuclease protection is used to assay the fate of decay intermediates. We have adapted the more sensitive and facile MBRACE assay (Lasham et al., Nucleic Acids Res 38: e19, 2010) to quantitatively monitor the decay process by detecting full-length β-globin and its decay intermediates.