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Identification of metabolite caps including FAD on the 5′ end of RNA has uncovered a previously unforeseen intersection between cellular metabolism and gene expression. To understand the function of FAD caps in cellular physiology, we characterised the proteins interacting with FAD caps in budding yeast. Here we demonstrate that highly conserved 5′-3′ exoribonucleases, Xrn1 and Rat1, physically interact with the RNA 5′ FAD cap and both possess FAD cap decapping (deFADding) activity and subsequently degrade the resulting RNA. Xrn1 deFADding activity was also evident in human cells indicating its evolutionary conservation. Furthermore, we report that the recently identified bacterial 5′-3′ exoribonuclease RNase AM also possesses deFADding activity that can degrade FAD-capped RNAs in vitro and in Escherichia coli cells. To gain a molecular understanding of the deFADding reaction, an RNase AM crystal structure with three manganese ions coordinated by a sulfate molecule and the active site amino acids was generated that provided details underlying hydrolysis of the FAD cap. Our findings reveal a general propensity for 5′-3′ exoribonucleases to hydrolyse and degrade RNAs with 5′ end noncanonical caps in addition to their well characterized 5′ monophosphate RNA substrates indicating an intrinsic property of 5′-3′ exoribonucleases.

Identification of metabolite caps including FAD on the 5’ end of RNA has uncovered a previously unforeseen intersection between cellular metabolism and gene expression. To understand the function of FAD caps in cellular physiology, we characterised the proteins interacting with FAD caps in budding yeast. Here we demonstrate that highly conserved 5’-3’ exoribonucleases, Xrn1 and Rat1, physically interact with the RNA 5’ FAD cap and both possess FAD cap decapping (deFADding) activity and subsequently degrade the resulting RNA. Xrn1 deFADding activity was also evident in human cells indicating its evolutionary conservation. Furthermore, we report that the recently identified bacterial 5’-3’ exoribonuclease RNase AM also possesses deFADding activity that can degrade FAD-capped RNAs in vitro and in E. coli cells. To gain a molecular understanding of the deFADding reaction, an RNase AM crystal structure with three manganese ions coordinated by a sulfate molecule and the active site amino acids was generated that provided details underlying hydrolysis of the FAD cap. Our findings reveal a general propensity for 5’-3’ exoribonucleases to hydrolyse and degrade RNAs with 5’ end noncanonical caps in addition to their well characterized 5’ monophosphate RNA substrates indicating an evolutionarily conserved intrinsic property of 5’-3’ exoribonucleases.

Regulation of enzymatic 5' decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5' decapping promotes accumulation of mRNAs into processing (P) bodies-membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7 (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7 inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of PPIP5Ks (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., PPIP5K KO), which elevates cellular 5-InsP7 levels by two- to threefold (i.e., within the physiological rheostatic range). The PPIP5K KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7 synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7 analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7 levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5′caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins—Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.

In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3′-FADP or CoA and fission yeast Rai1 with 3′-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.

We recently demonstrated that mammalian cells harbor nicotinamide adenine dinucleotide (NAD)-capped messenger RNAs that are hydrolyzed by the DXO deNADding enzyme. Here, we report that the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targeting different RNAs. The crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 A resolution provides insights into the molecular basis of the deNADding activity in the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells, and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to nutrient or environmental stress manifests changes in NAD-capped RNA levels that are selectively responsive to Nudt12 or DXO, respectively, indicating an association of deNADding to cellular metabolism. Structural and biochemical analyses identify Nudt12 as a novel mRNA deNADing enzyme in mammalian cells, which has a different substrate preference compared to the deNADing enzyme DXO.

The 5' N

The existence of non-canonical nicotinamide adenine diphosphate (NAD) 5’-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5’-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that still retains its 5’-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and in turn intramitochondrial NAD levels. Our findings uncover a functional role for mitochondrial NAD-capped RNAs as a reservoir to maintain overall NAD homeostasis. We propose NAD-capped RNAs function as a cistern for mitochondrial NAD with Xrn1 serving as a rheostat for NAD-capped RNAs.

The existence of non-canonical nicotinamide adenine diphosphate (NAD) 5′-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5′-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that predominantly still retains its 5′-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and may influence intramitochondrial NAD levels. Our findings uncover a contribution of mitochondrial NAD-capped RNAs in overall NAD regulation with the deNADding activity of Xrn1 fulfilling a central role.

Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth, and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP(7) (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP(7) inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of PPIP5Ks (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., PPIP5K KO), which elevates cellular 5-InsP(7) levels by two- to threefold (i.e., within the physiological rheostatic range). The PPIP5K KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP(7) synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP(7) analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP(7) levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

RNA 5′ cap structures comprising the metabolic effector nicotinamide adenine dinucleotide (NAD) have been identified in diverse organisms. Here we report a simple, two-step procedure to detect and quantitate NAD-capped RNA, termed “NAD-capQ.” By use of NAD-capQ we quantitate NAD-capped RNA levels in Escherichia coli, Saccharomyces cerevisiae, and human cells, and we measure increases in NAD-capped RNA levels in cells from all three organisms harboring disruptions in their respective “deNADding” enzymes. We further show that NAD-capped RNA levels in human cells respond to changes in cellular NAD concentrations, indicating that NAD capping provides a mechanism for human cells to directly sense and respond to alterations in NAD metabolism. Our findings establish NAD-capQ as a versatile, rapid, and accessible methodology to detect and quantitate 5′-NAD caps on endogenous RNA in any organism.

A hallmark of eukaryotic mRNAs has long been the 5'-end m7G cap. This paradigm was recently amended by recent reports that Saccharomyces cerevisiae and mammalian cells also contain mRNAs carrying a novel nicotinamide adenine dinucleotide (NAD+) cap at their 5'-end. The presence of an NAD+ cap on mRNA uncovers a previously unknown mechanism for controlling gene expression through nucleotide metabolite-directed mRNA turnover. In contrast to the m7G cap that stabilizes mRNA, the NAD+ cap targets RNA for rapid decay in mammalian cells through the DXO non-canonical decapping enzyme which removes intact NAD+ from RNA in a process termed 'deNADding'. This review highlights the identification of NAD+ caps, their mode of addition, and their functional significance in cells.

Recent studies have implicated long noncoding RNAs (lncRNAs) as regulators of many important biological processes. Here we report on the identification and characterization of a lncRNA, lnc13, that harbors a celiac disease-associated haplotype block and represses expression of certain inflammatory genes under homeostatic conditions. Lnc13 regulates gene expression by binding to hnRNPD, a member of a family of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). Upon stimulation, lnc13 levels are reduced, thereby allowing increased expression of the repressed genes. Lnc13 levels are significantly decreased in small intestinal biopsy samples from patients with celiac disease, which suggests that down-regulation of lnc13 may contribute to the inflammation seen in this disease. Furthermore, the lnc13 disease-associated variant binds hnRNPD less efficiently than its wild-type counterpart, thus helping to explain how these single-nucleotide polymorphisms contribute to celiac disease.

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

We report the chemical synthesis of a set of nicotinamide adenine dinucleotide (NAD) cap analogues containing chemical modifications that reduce their susceptibility to NAD-RNA-degrading enzymes. These analogues can be incorporated into transcripts in a similar way as NAD. Biochemical characterization of RNAs carrying these caps with DXO, NudC, and Nudt12 enzymes led to the identification of compounds that can be instrumental in unraveling so far unaddressed biological aspects of NAD-RNAs.

We recently demonstrated mammalian cells harbor NAD-capped mRNAs that are hydrolyzed by the DXO deNADding enzyme. Here we report the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targets different RNAs. Crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 Å resolution provides exquisite insights into the molecular basis of the deNADding activity within the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to metabolic stress manifests changes in NAD-capped RNA levels indicating an association between NAD-capped RNAs and cellular metabolism. Lastly, we show that the bacterial RppH protein also possesses deNADding activity toward NAD-capped RNA but not free NAD, revealing a third class of deNADding enzymes.

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex. SIGNIFICANCE STATEMENT The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by ingrowing axons from the thalamus regulates the combinatorial composition of ribosomal proteins in developing neocortex, which we term the “neocortical ribosome signature.” Thalamic WNT3 further regulates the specificity of mRNA translation and development of neurons and oligodendrocytes in the neocortex. This study advances our overall understanding of WNT signaling and the spatiotemporal regulation of mRNA translation in highly complex developing systems.

Recent studies showed that Rai1 and its homologs are a crucial component of the mRNA 5′-end capping quality control mechanism. They can possess RNA 5′-end pyrophosphohydrolase (PPH), decapping, and 5′-3′ exonuclease (toward 5′ monophosphate RNA) activities, which help to degrade mRNAs with incomplete 5′-end capping. A single active site in the enzyme supports these apparently distinct activities. However, each Rai1 protein studied so far has a unique set of activities, and the molecular basis for these differences are not known. Here, we have characterized the highly diverse activity profiles of Rai1 homologs from a collection of fungal organisms and identified a new activity for these enzymes, 5′-end triphosphonucleotide hydrolase (TPH) instead of PPH activity. Crystal structures of two of these enzymes bound to RNA oligonucleotides reveal differences in the RNA binding modes. Structure-based mutations of these enzymes, changing residues that contact the RNA but are poorly conserved, have substantial effects on their activity, providing a framework to begin to understand the molecular basis for the different activity profiles.

There are two known mRNA degradation pathways, 3′ to 5′ and 5′ to 3′. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3′ to 5′ exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5′ to 3′ degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects.

SUMMARYNucleoside-containing metabolites such as NAD+can be incorporated as “5′ caps” on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report “CapZyme-Seq,” a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-Seq with multiplexed transcriptomics, we determine efficiencies of NAD+capping byEscherichia coliRNAP for ~16,000 promoter sequences. The results define preferred transcription start-site (TSS) positions for NAD+capping and define a consensus promoter sequence for NAD+capping: HRRASWW (TSS underlined). By applying CapZyme-Seq toE. colitotal cellular RNA, we establish that sequence determinants for NCIN cappingin vivomatch the NAD+-capping consensus definedin vitro, and we identify and quantify NCIN-capped small RNAs. Our findings define the promoter-sequence determinants for NCIN capping with NAD+and provide a general method for analysis of NCIN cappingin vitroandin vivo.

A hallmark of eukaryotic mRNAs has long been the 5′ end m(7)G cap. This paradigm was recently amended by recent reports showing Saccharomyces cerevisiae and mammalian cells also contain mRNAs carrying a novel nicotinamide adenine dinucleotide (NAD(+)) cap at their 5′-end. The presence of an NAD(+) cap on mRNA uncovers a previously unknown mechanism for controlling gene expression through nucleoside metabolite-directed mRNA turnover. In contrast to the m(7)G cap that stabilizes mRNA, the NAD(+) cap targets RNA for rapid decay in mammalian cells through the DXO non-canonical decapping enzyme, which removes intact NAD(+) from RNA, in a process termed “deNADding”. This review highlights the identification of NAD(+) caps, their mode of addition, and functional significance in cells.

Messenger RNA precursors (pre-mRNAs) are produced as the nascent transcripts of RNA polymerase II (Pol II) in eukaryotes and must undergo extensive maturational processing, including 5'-end capping, splicing, and 3'-end cleavage and polyadenylation. This review will summarize the structural and functional information reported over the past few years on the large machinery required for the 3'-end processing of most pre-mRNAs, as well as the distinct machinery for the 3'-end processing of replication-dependent histone pre-mRNAs, which have provided great insights into the proteins and their subcomplexes in these machineries. Structural and biochemical studies have also led to the identification of a new class of enzymes (the DXO family enzymes) with activity toward intermediates of the 5'-end capping pathway. Functional studies demonstrate that these enzymes are part of a novel quality surveillance mechanism for pre-mRNA 5'-end capping. Incompletely capped pre-mRNAs are produced in yeast and human cells, in contrast to the general belief in the field that capping always proceeds to completion, and incomplete capping leads to defects in splicing and 3'-end cleavage in human cells. The DXO family enzymes are required for the detection and degradation of these defective RNAs.

Recently, we reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5' end-capped messenger RNAs (mRNAs). Here, we report that their mammalian homolog, Dom3Z (referred to as DXO), possesses pyrophosphohydrolase, decapping, and 5'-to-3' exoribonuclease activities. Surprisingly, we found that DXO preferentially degrades defectively capped pre-mRNAs in cells. Additional studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, a fact that contrasts with current understanding, and are also poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at a resolution of up to 1.5Å provide elegant insights into the catalytic mechanism and molecular basis for their three apparently distinct activities. Our data reveal a pre-mRNA 5' end capping quality control mechanism in mammalian cells, indicating DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5' end capping and subsequent pre-mRNA processing.

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 m7GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m7GMP and m7GDP. 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 m7GDP 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.

Summary Eukaryotic mRNAs generally possess a 5′ end N7 methyl guanosine (m 7 G) cap that promotes their translation and stability. However, mammalian mRNAs can also carry a 5′ end nicotinamide adenine dinucleotide (NAD + ) cap that, in contrast to the m 7 G cap, does not support translation but instead promotes mRNA decay. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD + caps, and cocrystal structures of DXO/Rai1 with 3′-NADP + illuminate the molecular mechanism for how the "deNADding" reaction produces NAD + and 5′ phosphate RNA. Removal of DXO from cells increases NAD + -capped mRNA levels and enables detection of NAD + -capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD + caps can be added to 5′-processed termini. Our findings establish NAD + as an alternative mammalian RNA cap and DXO as a deNADding enzyme modulating cellular levels of NAD + -capped RNAs. Collectively, these data reveal that mammalian RNAs can harbor a 5′ end modification distinct from the classical m 7 G cap that promotes rather than inhibits RNA decay.

Messenger RNAs transcribed by RNA polymerase II are modified at their 5'-end by the cotranscriptional addition of a 7-methylguanosine (m(7)G) cap. The cap is an important modulator of gene expression and the mechanism and components involved in its removal have been extensively studied. At least two decapping enzymes, Dcp2 and Nudt16, and an array of decapping regulatory proteins remove the m(7)G cap from an mRNA exposing the 5'-end to exonucleolytic decay. In contrast, relatively less is known about the decay of mRNAs that may be aberrantly capped. The recent demonstration that the Saccharomyces cerevisiae Rai1 protein selectively hydrolyzes aberrantly capped mRNAs provides new insights into the modulation of mRNA that lack a canonical m(7)G cap 5'-end. Whether an mRNA is uncapped or capped but missing the N7 methyl moiety, Rai1 hydrolyzes its 5'-end to generate an mRNA with a 5' monophosphate. Interestingly, Rai1 heterodimerizes with the Rat1 5'-3' exoribonuclease, which subsequently degrades the 5'-end monophosphorylated mRNA. Importantly, Rat1 stimulates the 5'-end hydrolysis activities of Rai1 to generate a 5'-end unprotected mRNA substrate for Rat1 and, in turn, Rai1 stimulates the activity of Rat1. The Rai1-Rat1 heterodimer functions as a molecular motor to detect and degrade mRNAs with aberrant caps and defines a novel quality control mechanism that ensures mRNA 5'-end integrity. The increase in aberrantly capped mRNA population following nutritional stress in S. cerevisiae demonstrates the presence of aberrantly capped mRNAs in cells and further reinforces the functional significance of the Rai1 in ensuring mRNA 5'-end integrity.

International audience; Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N-6,2'-O-dimethyladenosine (m(6)A(m)), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m(6)A(m) we find that m(6)A(m)-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m(6)A(m)-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m(6)A(m) is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m(6)A(m) rather than N-6-methyladenosine (m(6)A), and reduces the stability of m(6)A(m) mRNAs. Together, these findings show that the methylation status of m(6)A(m) in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

Removal of the 5' end cap is a critical determinant controlling mRNA stability and efficient gene expression. Removal of the cap is exquisitely controlled by multiple direct and indirect regulators that influence association with the cap and the catalytic step. A subset of these factors directly stimulate activity of the decapping enzyme, while others influence remodeling of factors bound to mRNA and indirectly stimulate decapping. Furthermore, the components of the general decapping machinery can also be recruited by mRNA-specific regulatory proteins to activate decapping. The Nudix hydrolase, Dcp2, identified as a first decapping enzyme, cleaves capped mRNA and initiates 5'-3' degradation. Extensive studies on Dcp2 led to broad understanding of its activity and the regulation of transcript specific decapping and decay. Interestingly, seven additional Nudix proteins possess intrinsic decapping activity in vitro and at least two, Nudt16 and Nudt3, are decapping enzymes that regulate mRNA stability in cells. Furthermore, a new class of decapping proteins within the DXO family preferentially function on incompletely capped mRNAs. Importantly, it is now evident that each of the characterized decapping enzymes predominantly modulates only a subset of mRNAs, suggesting the existence of multiple decapping enzymes functioning in distinct cellular pathways. WIREs RNA 2017, 8:e1379. doi: 10.1002/wrna.1379 For further resources related to this article, please visit the WIREs website.

Forkhead-box domain (Fox) containing family members are known to play a role in neocorticogenesis and have also been associated with disorders on the autism spectrum. Here we show that a single RNA-binding protein, Hu antigen R (HuR), dictates translation specificity of bound mRNAs and is sufficient to define distinct Foxp-characterized subpopulations of neocortical projection neurons. Furthermore, distinct phosphorylation states of HuR differentially regulate translation of Foxp mRNAs in vitro. This demonstrates the importance of RNA binding proteins within the framework of the developing brain and further confirms the role of mRNA translation in autism pathogenesis.

Summary Recent studies showed that Rai1 is a crucial component of the mRNA 5′-end capping quality control mechanism in yeast. The yeast genome encodes a weak homolog of Rai1, Ydr370C, but little is known about this protein. Here we report the crystal structures of Kluyveromyces lactis Ydr370C and the first biochemical and functional studies on this protein. The overall structure of Ydr370C is similar to Rai1. Ydr370C has robust decapping activity on RNAs with unmethylated caps but it has no detectable pyrophosphohydrolase activity. Unexpectedly, Ydr370C also possesses distributive, 5′-3′ exoribonuclease activity, and we propose the name Dxo1 for this novel eukaryotic enzyme with both decapping and exonuclease activities. Studies in yeast where both Dxo1 and Rai1 are disrupted reveal that mRNAs with incomplete caps are produced even under normal growth conditions, in sharp contrast to current understanding of the capping process.

The mammalian Dcp2 mRNA-decapping protein functions primarily on a subset of mRNAs in a transcript-specific manner. Here we show that Dcp2 is an important modulator of genes involved in the type I interferon (IFN) response, which is the initial line of antiviral innate immune response elicited by a viral challenge. Mouse embryonic fibroblast cells with reduced Dcp2 levels (Dcp2(β/β)) contained significantly elevated levels of mRNAs encoding proteins involved in the type I IFN response. In particular, analysis of a key type I IFN transcription factor, IFN regulatory factor 7 (IRF-7), revealed an increase in both IRF-7 mRNA and protein in Dcp2(β/β) cells. Importantly, the increase in IRF-7 mRNA within the background of reduced Dcp2 levels was attributed to a stabilization of the IRF-7 mRNA, suggesting that Dcp2 normally modulates IRF-7 mRNA stability. Moreover, Dcp2 expression was also induced upon viral infection, consistent with a role in attenuating the antiviral response by promoting IRF-7 mRNA degradation. The induction of Dcp2 levels following a viral challenge and the specificity of Dcp2 in targeting the decay of IRF-7 mRNA suggest that Dcp2 may negatively contribute to the innate immune response in a negative feedback mechanism to restore normal homeostasis following viral infection.

MicroRNAs (miRNAs) are endogenous single-stranded RNA molecules of about 21 nucleotides in length that are fundamental post-transcriptional regulators of gene expression. Although the transcriptional and processing events involved in the generation of miRNAs have been extensively studied, very little is known pertaining to components that regulate the stability of individual miRNAs. All RNAs have distinct inherent half-lives that dictate their level of accumulation and miRNAs would be expected to follow a similar principle. Here we demonstrate that although most miRNA appear to be stable, like mRNAs, miRNAs possess differential stability in human cells. In particular, we found that miR-382, a miRNA that contributes to HIV-1 provirus latency, is unstable in cells. To determine the region of miR-382 responsible for its rapid decay, we developed a cell-free system that recapitulated the observed cell-based-regulated miR-382 turnover. The system utilizes in vitro-processed mature miRNA derived from pre-miRNA and follows the decay of the processed miRNA. Using this system, we demonstrate that instability of miR-382 is driven by sequences outside its seed region and required the 3′ terminal seven nucleotides where mutations in this region increased the stability of the RNA. Moreover, the exosome 3′–5′ exoribonuclease complex was identified as the primary nuclease involved in miR-382 decay with a more modest contribution by the Xrn1 and no detectable contribution by Xrn2. These studies provide evidence for an miRNA element essential for rapid miRNA decay and implicate the exosome in this process. The development of a biochemically amendable system to analyze the mechanism of differential miRNA stability provides an important step in efforts to regulate gene expression by modulating miRNA stability.

The 5'-->3' exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference. XRN2, or Rat1 in yeast, functions primarily in the nucleus and also has an important role in transcription termination by RNA polymerase II (refs 7-14). Rat1 exoribonuclease activity is stimulated by the protein Rai1 (refs 15, 16). Here we report the crystal structure at 2.2 A resolution of Schizosaccharomyces pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homologue Dom3Z alone at 2.0 A resolution. The structures reveal the molecular mechanism for the activation of Rat1 by Rai1 and for the exclusive exoribonuclease activity of Rat1. Biochemical studies confirm these observations, and show that Rai1 allows Rat1 to degrade RNAs with stable secondary structure more effectively. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N terminus) domain-containing nucleases. Unexpectedly, we identified a large pocket in Rai1 and Dom3Z that contains highly conserved residues, including three acidic side chains that coordinate a divalent cation. Mutagenesis and biochemical studies demonstrate that Rai1 possesses pyrophosphohydrolase activity towards 5' triphosphorylated RNA. Such an activity is important for messenger RNA degradation in bacteria, but this is, to our knowledge, the first demonstration of this activity in eukaryotes and suggests that Rai1/Dom3Z may have additional important functions in RNA metabolism.

The DcpS scavenger decapping enzyme carries out the final step of the 3' to 5' end mRNA decay pathway by clearing the cell of residual cap structure that would otherwise accumulate. Its ability to modulate cap structure accumulation in a cell implies that DcpS could influence the pool of available cap-binding proteins and in turn impact their downstream functions. Since the 5' cap and corresponding cap-binding proteins are integral components of various aspects of mRNA metabolism including pre-mRNA splicing, nuclear export, translation and decay, DcpS has been proposed as a possible regulator of these processes. There is now increasing evidence that DcpS could impact at least some of these events. Indeed, DcpS can regulate the cap-dependent first intron pre-mRNA splicing in mammalian cells and a DcpS ortholog is important for 5' to 3' exonucleolytic activity in yeast. Moreover, the nucleocytoplasmic shuttling property of DcpS indicates that it could function as a regulator of cap-dependent processes in both cellular compartments by buffering cap structure concentrations and the resulting availability of cap-binding proteins in a cell. In this review, we discuss known and potential roles of DcpS in mRNA metabolism.

The human scavenger decapping enzyme, DcpS, functions to hydrolyze the resulting cap structure following cytoplasmic mRNA decay yet is, surprisingly, a nuclear protein by immunofluorescence. Here, we show that DcpS is a nucleocytoplasmic shuttling protein that contains separable nuclear import and Crm-1-dependent export signals. We postulated that the presence of DcpS in both cellular compartments and its ability to hydrolyze cap structure may impact other cellular events dependent on cap-binding proteins. An shRNA-engineered cell line with markedly diminished DcpS levels led to a corresponding reduction in cap-proximal intron splicing of a reporter minigene and endogenous genes. The impaired cap catabolism and resultant imbalanced cap concentrations were postulated to sequester the cap-binding complex (CBC) from its normal splicing function. In support of this explanation, DcpS efficiently displaced the nuclear cap-binding protein Cbp20 from cap structure, and complementation with Cbp20 reversed the reduced splicing, indicating that modulation of splicing by DcpS is mediated through Cbp20. Our studies demonstrate that the significance of DcpS extends beyond its well-characterized role in mRNA decay and involves a broader range of functions in RNA processing including nuclear pre-mRNA splicing.

Decapping is a critical step in the control of gene expression and is regulated by both positive and negativetransfactors. Less is known aboutciselements that promote decapping. In plants, following microRNA (miRNA)-directed cleavage of an mRNA, a uridine tract can be added onto the exposed 3′ end of the resulting 5′ fragment, which can promote 5′ end decay. We now demonstrate that in mammalian cell extract, addition of five uridine residues to the 3′ end of an RNA (U5) promotes decapping relative to an RNA lacking the uridines (U0). Although the decapping stimulation observed in extract required hDcp2, recombinant hDcp2 was unable to support differential decapping of the U0and U5RNAs, indicating that the stimulation was likely due to an indirect recruitment of hDcp2 to the RNA. Consistent with the promotion of 5′ end decapping by the uridine tract, affinity purification with the U5RNA revealed the presence of a decapping subcomplex at least consisting of hDcp2, Dcp1a, Edc4, LSm1, and LSm4 that were specifically bound to the U5RNA but not the U0RNA. In addition to promoting decapping, the U-tract stabilized the 3′ end of the RNA by preventing 3′ to 5′ exonucleolytic decay to ensure 5′ 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 5′ end decapping to expedite mRNA decay.

Messenger RNA (mRNA) decay utilizes both exoribonucleolytic and endoribonucleolytic enzymes where the latter are generally more prone to be transcript-specific. An erythroid-enriched endoribonuclease, ErEN, can destabilize the alpha-globin mRNA through directing a site-specific cleavage within the 3' untranslated region (3' UTR) both in vitro and in vivo. ErEN activity is sequence- and/or local structure-specific as the minimal recognition/cleavage sequence can be conferred onto a heterologous RNA and mutations at the cleavage site immunize the mRNA from ErEN hydrolysis. Interestingly, the ErEN cleavage activity is regulated by an mRNA stability complex (alpha-complex). An interaction between the alpha-complex and the poly(A)-binding protein (PABP) accentuates alpha-complex binding to a region overlapping the ErEN cleavage site and further prevents premature ErEN-mediated decay. At present the identity of ErEN remains elusive, yet its identification will provide mechanistic and functional insights into the general processes of endoribonuclease-mediated mRNA turnover and erythropoiesis.

Removal of the 5′-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin β6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin β6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin β6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.

Two major decapping enzymes are involved in the decay of eukaryotic mRNA, Dcp2 and DcpS. Despite the detection of robust DcpS decapping activity in cell extract, minimal to no decapping is detected from human Dcp2 (hDcp2) in extract. We now demonstrate that one reason for the lack of detectable hDcp2 activity in extract is due to the presence of inhibitory trans factor(s). Furthermore, we demonstrate that a previously identified testis-specific protein of unknown function implicated in nonspecific X-linked mental retardation, VCX-A, can function as an inhibitor of hDcp2 decapping in vitro and in cells. VCX-A is a noncanonical cap-binding protein that binds to capped RNA but not cap structure lacking an RNA. Its cap association is enhanced by hDcp2 to further augment the ability of VCX-A to inhibit decapping. Our data demonstrate that VCX-A can regulate mRNA stability and that it is an example of a tissue-specific decapping regulator.

Removal of the mRNA 5′ cap is an important step in the regulation of mRNA stability. mRNAs are degraded by at least two distinct exonucleolytic decay pathways, one from the 5′ end, and the second from the 3′ end. Two major cellular decapping enzymes have been identified, and each primarily functions in one of the two decay pathways. The Dcp2 decapping enzyme utilizes capped mRNA as substrate and hydrolyses the cap to release m7GDP (N7-methyl GDP), while a scavenger decapping enzyme, DcpS, utilizes cap dinucleotides or capped oligonucleotides as substrate and releases m7GMP (N7-methyl GMP). In this review, we will highlight the function of different decapping enzymes and their role in mRNA turnover.

A unique characteristic of eukaryotic mRNA is the N7-methylated guanosine cap structure that is cotranscriptionally added to the 5′ terminus of nascent RNA (40). The cap serves to protect the 5′ end of the mRNA from exoribonucleolytic degradation (49). It also functions in the transport of mature mRNA from the nucleus to the cytoplasm (18, 20), in translation initiation (12), and in pre-mRNA splicing (24). The cap is bound by distinct cap-binding proteins in each cellular compartment. A heterodimeric complex of CBC20 and CBC80 binds the cap in the nucleus (19), while eIF4E, the translation initiation factor, associates with the cap in the cytoplasm (12). In addition to these major cap binding proteins, several other factors have also been demonstrated to bind the mRNA cap, including the mammalian poly(A)-binding protein (22), the cold shock protein YB-1 (10), and the scavenger decapping protein (28, 37). Decay of mRNA is not a randomized process and proceeds through at least two major decay pathways, both of which are initiated by removal of the poly(A) tail (31). Following deadenylation, the mRNA is decapped and degraded by a 5′ to 3′ exonucleolytic activity in the 5′ decay pathway. In the 3′ decay pathway, the mRNA is continuously degraded from the 3′ end following deadenylation, generating an m7GpppN cap dinucleotide that is hydrolyzed by a scavenger decapping activity (27, 48). Each mRNA decay pathway utilizes a unique decapping activity with a distinct substrate specificity (7). In the 5′ pathway, both the yeast and human Dcp2 proteins utilize capped mRNA as a substrate and hydrolyze the cap structure to generate m7GDP and RNA with a monophosphate at its 5′ end (29, 41, 43, 47). The exposed 5′ end of the RNA is degraded by Xrn1p, the 5′ to 3′ exoribonuclease, leading to rapid degradation of the RNA body (4, 17, 25). A scavenger decapping activity termed DcpS in mammals and Dcs1p in budding yeast functions in the final step of the 3′ decay pathway. This activity hydrolyzes the residual cap structure following 3′ to 5′ exonucleolytic decay by the exosome complex to release m7GMP and nucleotide diphosphate (27, 48). Characterization of scavenger decapping activity indicates its strong preference for binding and hydrolyzing cap structure linked to an RNA of less than 10 nucleotides in mammals and 3 nucleotides in Caenorhabditis elegans (6, 27, 28). Recent structural analysis of DcpS reveals that it can form either an asymmetric dimer bound to two cap dinucleotides (13) or a symmetric dimer in the ligand-free protein (5). The dimers contain distinct amino-terminal and carboxyl-terminal domains separated by a hinge region (13). The structure indicates that the N terminus can flip back and forth, alternating on each side from a productive closed to a nonproductive open conformation (13). An interesting property of DcpS is its almost exclusive utilization of the residual cap dinucleotide following 3′ exonucleolytic decay of the RNA (27, 28). This substrate specificity is due in part to a higher affinity of DcpS for the cap structure (28) and more significantly to both entropic and steric constraints in the formation of a closed decapping-competent complex in the presence of an mRNA moiety on the cap (13). Saccharomyces cerevisiae contains two proteins termed Dcs1p and Dcs2p that are equally homologous to the human DcpS. Curiously, only Dcs1p possesses intrinsic decapping activity analogous to that of DcpS (27). The enzymatic activity and substrate specificity for Dcs2p remain unknown, although its ability to heterodimerize with Dcs1p suggests that it might be a modulator of Dcs1p decapping activity (30). Dcs1p is a member of the histidine triad (HIT) family of nucleotide binding proteins and contains the characteristic HIT motif (His-X-His-X-His-X, where X is a hydrophobic amino acid) which is required for its cap hydrolysis activity (27). By virtue of its ability to hydrolyze the cap dinucleotide (27), DcpS and Dcs1p are postulated to function during the terminal phase of mRNA decay. Here we demonstrate that Dcs1p also functions to impact the 5′ mRNA decay pathway.

Hydrolysis of the mRNA cap plays a pivotal role in initiating and completing mRNA turnover. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We describe here the characterization of nematode Dcp1 and Dcp2 proteins. Dcp1 was inactive in vitro on both free cap and capped RNA and did not significantly enhance Dcp2 activity. Nematode Dcp2 is an RNA-decapping protein that does not bind cap and is not inhibited by cap analogs but is effectively inhibited by competing RNA irrespective of RNA sequence and cap. Nematode Dcp2 activity is influenced by both 5' end sequence and its context. The trans-spliced leader sequence on mRNAs reduces Dcp2 activity approximately 10-fold, suggesting that 5'-to-3' turnover of trans-spliced RNAs may be regulated. Nematode Dcp2 decaps both m(7)GpppG- and m(2,2,7)GpppG-capped RNAs. Surprisingly, both budding yeast and human Dcp2 are also active on m(2,2,7)GpppG-capped RNAs. Overall, the data suggest that Dcp2 activity can be influenced by both sequence and context and that Dcp2 may contribute to gene regulation in multiple RNA pathways, including monomethyl- and trimethylguanosine-capped RNAs.