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.

Decapping is a critical step in the control of gene expression and is regulated by both positive and negative trans factors. Less is known about cis elements that promote decapping. In plants, following microRNA (miRNA)-directed cleavage of an mRNA, a uridine tract can be added onto the exposed 39 end of the resulting 59 fragment, which can promote 59 end decay. We now demonstrate that in mammalian cell extract, addition of five uridine residues to the 39 end of an RNA (U 5 ) promotes decapping relative to an RNA lacking the uridines (U 0 ). Although the decapping stimulation observed in extract required hDcp2, recombinant hDcp2 was unable to support differential decapping of the U 0 and U 5 RNAs, indicating that the stimulation was likely due to an indirect recruitment of hDcp2 to the RNA. Consistent with the promotion of 59 end decapping by the uridine tract, affinity purification with the U 5 RNA revealed the presence of a decapping subcomplex at least consisting of hDcp2, Dcp1a, Edc4, LSm1, and LSm4 that were specifically bound to the U 5 RNA but not the U 0 RNA. In addition to promoting decapping, the U-tract stabilized the 39 end of the RNA by preventing 39 to 59 exonucleolytic decay to ensure 59 end directional degradation. These data suggest that following post-transcriptional oligo uridylation of an mRNA or mRNA fragment, the U-tract has the capacity to specifically stimulate 59 end decapping to expedite mRNA decay.