A kinetic assay to monitor RNA decapping under single- turnover conditions.

The stability of all RNA polymerase II transcripts depends on the 5'-terminal cap structure. Removal of the cap is a prerequisite for 5' to 3'-decay and is catalyzed by distinct cellular and viral decapping activities. Over the past decade, several decapping enzymes have been characterized through functional and structural studies. An emerging theme is that function is regulated by protein interactions; however, in vitro assays to dissect the effects on enzyme activity are unavailable. Here we present a kinetic assay to monitor decapping by the heterodimeric yeast Dcp1/Dcp2 complex. Kinetic constants related to RNA binding and the rate of the catalytic step can be determined with recombinant enzyme and cap-radiolabeled RNA substrate, allowing substrate specificity and the role of activating factors to be firmly established.

mRNA decapping is promoted by an RNA-binding channel in Dcp2.

Cap hydrolysis by Dcp2 is a critical step in several eukaryotic mRNA decay pathways. Processing requires access to cap-proximal nucleotides and the coordinated assembly of a decapping mRNP, but the mechanism of substrate recognition and regulation by protein interactions have remained elusive. Using NMR spectroscopy and kinetic analyses, we show that yeast Dcp2 resolves interactions with the cap and RNA body using a bipartite surface that forms a channel intersecting the catalytic and regulatory Dcp1-binding domains. The interaction with cap is weak but specific and requires binding of the RNA body to a dynamic interface. The catalytic step is stimulated by Dcp1 and its interaction domain, likely through a substrate-induced conformational change. Thus, activation of the decapping mRNP is restricted by access to 5'-proximal nucleotides, a feature that could act as a checkpoint in mRNA metabolism.