Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex

Abstract

Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5 ′ m 7 G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m 7 GpppN) and m 7 GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m 7 G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m 7 Gp S ppp S m 7 G, inhibited Dcp1/2 20 times more efficiently than m 7 GpppN or m 7 GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m 7 Gp S ppp S m 7 G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m 7 Gp S ppp S m 7 G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors.

Structural basis of mRNA-cap recognition by Dcp1–Dcp2

“…Mutation of Y92 results in a ~5-fold defect in decapping in vivo . 32 Interestingly, Y92 resonances are not perturbed by addition of m 7 GDP or two-headed cap analog in NMR titration experiments with the isolated Dcp2 regulatory domain, 37,40 suggesting conformational changes in the hinge region and its coupling with Nudix domain motions, are critical for cap recognition. Upon cap binding, conserved residue K93 moves from a flexible linker to a structured helix and likely makes multiple hydrogen bond contacts with the phosphate chain of cap; its mutation results in modest decapping defects both in vitro and in vivo .…”

“…The two-headed cap analog consists of two m 7 G nucleotides linked by a tetraphosphate bridge, which binds 20-fold more tightly than native cap and is readily hydrolyzed by Dcp2 as the cap analog alone, or when incorporated at the 5′ end of RNA. 40 Binding of this cap analog induces a significant conformational change in Dcp2 within the crystal, consisting primarily of a 25° rotation of the Nudix domain away from Dcp1, relative to the apo structure (Fig. 1c, Supplementary Video 1).…”

“…40,41 We sought to use this tight-binding cap analog, in combination with Edc or PNRC coactivators, as tools to trap the substrate-bound, catalytically-active conformation of the Dcp1–Dcp2 decapping complex. Here we show that binding of cap analog to a PNRC2–Dcp1–Dcp2 complex induces a heretofore unknown conformation of Dcp2 that reveals a composite substrate binding site composed of residues on the regulatory and Nudix domains.…”

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

“…6) [63]. 32 P-labeled capped RNA oligonucleotides were used to evaluate the susceptibility of various synthetic cap structures modified in the triphosphate bridge (discussed in more detail below) to Dcp2 [64,65] and to screen potential inhibitors of Dcp2-catalyzed decapping [66]. If short capped RNA oligonucleotides (up to 50 nt) were used as Dcp2 substrates, the electrophoretic resolution of capped and decapped RNAs could be performed directly on sequencing gels.…”

“…Another example of the usefulness of MCl 2 -mediated coupling reactions involving P-imidazolides is the synthesis of a symmetrical two-headed tetraphosphate cap analog [91], which was found to be a potent inhibitor of Dcp2 decapping enzyme acting as an mRNA 5 0 cap mimic [27,66]. Owing to the symmetry of the final compound, it was possible to perform two coupling reactions in one synthetic step, starting with P 1 ,P 2 -diimidazolyl-pyrophosphate and two equivalents of 7-methylguanosine 5 0 -phosphorothioate (Fig.…”