Application of the Invader RNA Assay to the Polarity of Vertebrate mRNA Decay

The inability of structural elements within a reporter mRNA to impede processive decay by the major 5′ and 3′ exonucleases has been a major obstacle to understanding mechanisms of vertebrate mRNA decay. We present here a new approach to this problem focused on quantifying the decay of individual portions of a reporter mRNA. Our approach entails two parts. The first involves the use of a regulated promoter, such as one controlled by tetracycline (tet), to allow reporter gene transcription to be turned off when needed. Cells stably expressing the tet repressor protein are transiently or stably transfected with tet-regulated beta-globin genes in which the sequence element under study is cloned into the 3′-UTR. The second involves the quantification of beta-globin mRNA using the Invader RNA assay, a sensitive and quantitative approach that relies on signal amplification instead of target amplification. Because the Invader RNA assay does not depend on downstream primer binding, the use of multiple probes across the reporter beta-globin mRNA allows for quantification of the decay of individual portions of the mRNA independent of events acting at other sites.