7-Methylguanosine Diphosphate (m⁷GDP) Is Not Hydrolyzed but Strongly Bound by Decapping Scavenger (DcpS) Enzymes and Potently Inhibits Their Activity

Abstract

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3′→5′ mRNA decay. Some previous studies suggested that both m7GpppG and m7GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m7GDP (7-methylguanosine diphosphate) and m32,2,7GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5′→3′ mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode and yeast). Furthermore, whereas mononucleoside diphosphates (m7GDP and m32,2,7GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m7GTP and m32,2,7GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m7GTP and m32,2,7GTP are cleaved at a slower rate than their corresponding dinucleotides (m7GpppG and m32,2,7GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m7GDP, they have a high binding affinity for m7GDP and m7GDP potently inhibits DcpS hydrolysis of m7GpppG, suggesting that m7GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m7GDP in mRNA metabolic pathways, due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Multiple Nudix family proteins possess mRNA decapping activity

“…3), which may account for why only m 7 GMP was detected as the decapping product for Nudt15 in Figure 1A. Moreover, we were unable to detect a nucleoside diphosphatase activity for DcpS, which has been reported to convert m 7 Gpp to m 7 Gp (van Dijk et al 2003) and is consistent with a more recent report that demonstrated although DcpS avidly binds m 7 Gpp, it does not hydrolyze this substrate (Wypijewska et al 2012).…”

Identification of Drosophila and Human 7-Methyl GMP-specific Nucleotidases

“…In fact, m 7 GDP is converted to m 7 GMP in extracts of different cells (23). DcpS has been reported to be responsible for this reaction (23,24), but this has not been confirmed by others (12,25,26). Regardless, m 7 GMP appears to be a universal intermediate of cap degradation but probably not its final product.…”

Effect of different N7 substitution of dinucleotide cap analogs on the hydrolytic susceptibility towards scavenger decapping enzymes (DcpS)

“…Decapping scavengers: human DcpS (HsDcpS), C. elegans DcpS (CeDcpS) and A. suum DcpS (AsDcpS) were expressed in Escherichia coli and purified as His-tagged proteins by affinity chromatography using Ni-NTA agarose under native conditions. To obtain homogeneous fractions of DcpS, recombinant enzymes were further purified by gel filtration through a Pharmacia Superdex-200 column (GE Healthcare Bioscience AB) connected to AKTA FPLC system (Pharmacia-Biotech) [14]. The concentration of cap analogs and DcpS enzymes were estimated from their molar absorption coefficients [14,22].…”

“…However, the hydrolytic activity and binding affinity have been well characterized for human and Caenorhabditis elegans DcpS, indicating that these two enzymes differ in substrate length and capacity to hydrolyze trimethyleted cap analogs [14,15]. For the specific cap recognition and efficient degradation by DcpS, the following structural features are required: (1) the positive charge at the N7 position of guanine moiety, (2) 2 0 OH and 3 0 OH groups in the ribose ring of 7-methylguanosine, (3) at least triphosphate groups in the phosphate bridge [15].…”