Application of the Invader RNA assay to the polarity of vertebrate mRNA decay

Abstract

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.

Fig. 1

Outline of the Invader RNA assay. The steps involved in the Invader RNA assay are shown. The stacking oligo abuts the probe oligo on the target mRNA and the invader oligo overlaps the junction of the probe oligo and the flap by 1 nucleotide. Cleavase enzyme cuts within the distorted structure formed by this complex to generate flaps in direct proportion to the concentration of target mRNA. These serve as the invader in a second reaction in which an oligo bearing a fluorescent tag and a quencher are separated by the cleavage site in a complex assembled on a generic second reaction template (SRT). Fluorescence is generated in direct proportion to the concentration of flaps generated in the first reaction.

Fig. 2

Standard curve for an optimal Invader set. This is a standard curve for the Invader set directed against exon 1 of human β-globin mRNA that is described in this chapter. Note that the line goes through zero and shows a linear increase in light output with increasing amount of in vitro transcript.

Fig. 3

Standard curve for an Invader set that shows no relationship to input RNA. The Invader set examined in this standard curve lies just upstream of the exon 1 set shown in Fig. 2. However no change in light output is seen for this probe set when increasing amounts of the same RNA are added to the reaction.

Fig. 4

Standard curve for an Invader set that does not go through zero. This is a standard curve for an Invader set in which light output is recorded with no input RNA. Although the amount of light detected increases with increasing transcript this cannot be used.