Biochemical analysis of the mRNA scavenger decapping enzymes

The modulation of mRNA degradation is an essential determining point for regulation of gene expression. Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNAs. The scavenger mRNA decapping enzyme, DcpS, hydrolyzes the cap structure, which is the decay product in the 3'-5' mRNA degradation pathway. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m7GpppN to release m7Gp and ppN. We have carried out a biochemical characterization of the DcpS enzyme and demonstrated that, unlike other HIT family members, DcpS requires both the core HIT fold at the C-terminus and a segment of N terminus for cap binding and hydrolysis. To further examine the cellular function of DcpS, we tested the impact of DcpS on cap-dependent translation. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure in the presence of eIF4E, an essential cap-binding translation initiation factor. Furthermore, we demonstrated that the relative cap-dependent translation was inhibited by 40% in the DcpS knockdown cells and was partially restored by DcpS complementation. These results strongly suggest that DcpS functions to prevent the accumulation of residual cap structure that would otherwise trap eIF4E and interfere with cap-dependent translational events. Structural analysis of DcpS revealed that it is a dimeric protein with a distinct N terminal domain and a C terminal domain, linked by a flexible hinge region, which led to a proposed dynamic decapping model, where the N terminus flips back and forth during the process of hydrolysis. To gain more insights into the decapping mechanism at the subunit level, we analyzed the kinetics of DcpS decapping and demonstrated that its decapping was negatively regulated under multiple turnover conditions, due to an allosteric conformational change caused by the excess amount of substrate. Our data have provided mechanistic details in terms of hydrolysis as well as the insights into the regulation of decapping in cells.Ph.D.Includes bibliographical references (p. 108-125)