Multiple Nudix family proteins possess mRNA decapping activity

Abstract

RNA decapping is an important contributor to gene expression and is a critical determinant of mRNA decay. The recent demonstration that mammalian cells harbor at least two distinct decapping enzymes that preferentially modulate a subset of mRNAs raises the intriguing possibility of whether additional decapping enzymes exist. Because both known decapping proteins, Dcp2 and Nudt16, are members of the Nudix hydrolase family, we set out to determine whether other members of this family of proteins also contain intrinsic RNA decapping activity. Here we demonstrate that six additional mouse Nudix proteins-Nudt2, Nudt3, Nudt12, Nudt15, Nudt17, and Nudt19-have varying degrees of decapping activity in vitro on both monomethylated and unmethylated capped RNAs. The decapping products from Nudt17 and Nudt19 were analogous to Dcp2 and predominantly generated m 7 GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m 7 GMP and m 7 GDP. Interestingly, all six Nudix proteins as well as both Dcp2 and Nudt16 could hydrolyze the cap of an unmethylated capped RNA, indicating that decapping enzymes may be less constrained for the presence of the methyl moiety. Investigation of Saccharomyces cerevisiae Nudix proteins revealed that the yeast homolog of Nudt3, Ddp1p, also possesses decapping activity in vitro. Moreover, the bacterial Nudix pyrophosphohydrolase RppH displayed RNA decapping activity and released m 7 GDP product comparable to Dcp2, indicating that decapping is an evolutionarily conserved activity that preceded mammalian cap formation. These findings demonstrate that multiple Nudix family hydrolases may function in mRNA decapping and mRNA stability.

Nudt3 is an mRNA decapping enzyme that modulates cell migration

“…1A). The catalytic activity of Nudt3 has been shown to hydrolyze two primary substrates in vitro, dinucleoside polyphosphates (Ap n A) (McLennan 2006) and capped mRNA (Song et al 2013). To distinguish which of these activities contributed to Nudt3's role in up-regulating specific mRNA levels, we determined the mRNA expression in cells lacking Nudt4.…”

“…To determine whether the catalytic activity of Nudt3 was necessary, Nudt3 KD cells were complemented with Flagtagged shRNA-resistant wild-type or a catalytically inactive mutant (Nudt3 EE/QQ ) harboring glutamine substitutions for two glutamic acid metal coordination residues that abolish Nudt3 decapping activity (Song et al 2013). Exogenous proteins were expressed at levels comparable to endogenous Nudt3 (Fig.…”

“…The Nudix family of proteins are evolutionarily conserved with 22 Nudix proteins identified in the human genome. Although Dcp2 and Nudt16 are the only known mammalian Nudix proteins that have been reported to decap RNA in cells, six additional mammalian Nudix proteins possess decapping activity at least in vitro (Song et al 2013). Nudt2, Nudt3, Nudt12, and Nudt15 hydrolyze methylated capped RNA with the activity comparable to Dcp2 or Nudt16, while Nudt17 and Nudt19 possess modest decapping activity in vitro (Song et al 2013).…”

“…Six Nudix proteins are present in yeast Saccharomyces cerevisiae. However, other than Dcp2, only one of the five Nudix proteins, Ddp1p, demonstrated robust decapping activity in vitro (Song et al 2013). Interestingly, Ddp1p appears to be the homolog of mammalian Nudt3 suggesting that Nudt3 and Ddp1p may be evolutionarily conserved decapping enzymes.…”

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

“…There are no reports indicating that scavenger decapping enzymes are able to hydrolyze the m 7 G cap structure attached to an RNA chain longer than several nucleotides (Milac et al 2014). Recent evidence suggests that in mammals, many Nudix hydrolase family enzymes (Nudt2,3,12,15,16,17) may have decapping activity in addition to Dcp2 (Taylor and Peculis 2008;Song et al 2010Song et al , 2013Li and Kiledjian 2011;Lu et al 2011). Finally, the DXO family of enzymes possess pyrophosphohydrolase, decapping, and 5…”