Analysis of mRNA decapping.

The modulation of mRNA decay is a critical determinant in the regulation of gene expression. mRNAs in eukaryotes are primarily degraded by two major exonucleolytic pathways: the 5' to 3'-and the 3' to 5'-pathways, both of which are initiated by removal of the polyadenylated (poly(A)) tail. Hydrolysis of the 5'-cap structure, termed decapping, is a key step in the demise of mRNA. Two major decapping enzymes with distinct activities and substrate requirements have been identified. Dcp2 hydrolyzes the cap structure on an intact mRNA in the 5' to 3'-decay pathway; Dcp2 scavenges the residual cap oligonucleotide resulting from the 3' to 5'-decay pathway, as well as hydrolyzes the decapping product generated by Dcp2. In this chapter, we describe the methods for monitoring Dcp2 and DcpS decapping activities of bacterially expressed and endogenous human decapping enzymes.