Functional analysis of mRNA scavenger decapping enzymes

Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNA. Two major decapping enzymes have been identified. The hDcp2 protein catalyzes hydrolysis of the 5' cap linked to an RNA moiety, whereas the scavenger decapping enzyme, DcpS, functions on a cap structure lacking the RNA moiety. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m7GpppN. HIT proteins are homodimeric and contain two conserved 100-amino-acid HIT fold domains with independent active sites that are each sufficient to bind and hydrolyze cognate substrates. We carried out a functional characterization of the DcpS enzyme and demonstrate that unlike previously described HIT proteins, DcpS is a modular protein that requires both the core HIT fold at the carboxyl-terminus and sequences at the amino-terminus of the protein for cap binding and hydrolysis. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure even in the presence of excess eIF4E, implying that DcpS could function to alleviate the accumulation of complexes between eIF4E and cap structure that would otherwise accumulate following mRNA decay. Using immunofluorescence microscopy, we demonstrate that DcpS is predominantly a nuclear protein, with low levels of detected protein in the cytoplasm. Furthermore, analysis of the endogenous hDcp2 protein reveals that in addition to the cytoplasmic foci, it is also present in the nucleus. These data reveal that both decapping enzymes are contained in the nuclear compartment, indicating that they may fulfill a greater function in the nucleus than previously appreciated.     

Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping

Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3'-5' mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG-DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release.     

Nematode m7GpppG and m3(2,2,7)GpppG decapping: activities in Ascaris embryos and characterization of C. elegans scavenger DcpS

A spliced leader contributes the mature 5'ends of many mRNAs in trans-splicing organisms. Trans-spliced metazoan mRNAs acquire an m3(2,2,7)GpppN cap from the added spliced leader exon. The presence of these caps, along with the typical m7GpppN cap on non-trans-spliced mRNAs, requires that cellular mRNA cap-binding proteins and mRNA metabolism deal with different cap structures. We have developed and used an in vitro system to examine mRNA degradation and decapping activities in nematode embryo extracts. The predominant pathway of mRNA decay is a 3' to 5' pathway with exoribonuclease degradation of the RNA followed by hydrolysis of resulting mRNA cap by a scavenger (DcpS-like) decapping activity. Direct decapping of mRNA by a Dcp1/Dcp2-like activity does occur, but is approximately 15-fold less active than the 3' to 5' pathway. The DcpS-like activity in nematode embryo extracts hydrolyzes both m7GpppG and m3(2,2,7)GpppG dinucleoside triphosphates. The Dcp1/Dcp2-like activity in extracts also hydrolyzes these two cap structures at the 5' ends of RNAs. Interestingly, recombinant nematode DcpS differs from its human ortholog in its substrate length requirement and in its capacity to hydrolyze m3(2,2,7)GpppG.     

Mechanistic and kinetic analysis of the DcpS scavenger decapping enzyme

Decapping is an important process in the control of eukaryotic mRNA degradation. The scavenger decapping enzyme DcpS functions to clear the cell of cap structure following decay of the RNA body by catalyzing the hydrolysis of m(7)GpppN to m(7)Gp and ppN. Structural analysis has revealed that DcpS is a dimeric protein with a domain-swapped amino terminus. The protein dimer contains two cap binding/hydrolysis sites and displays a symmetric structure with both binding sites in the open conformation in the ligand-free state and an asymmetric conformation with one site open and one site closed in the ligand-bound state. The structural data are suggestive of a dynamic decapping mechanism where each monomer could alternate between an open and closed state. Using transient state kinetic studies, we show that both the rate-limiting step and rate of decapping are regulated by cap substrate. A regulatory mechanism is established by the intrinsic domain-swapped structure of the DcpS dimer such that the decapping reaction is very efficient at low cap substrate concentrations yet regulated with excess cap substrate. These data provide biochemical evidence to verify experimentally a dynamic and mutually exclusive cap hydrolysis activity of the two cap binding sites of DcpS and provide key insights into its regulation.     

Decapping Scavenger (DcpS) enzyme: Advances in its structure,activity and roles in the cap-dependent mRNA metabolism

Decapping Scavenger (DcpS) enzyme rids eukaryotic cells of short mRNA fragments containing the 5′ mRNA cap structure, which appear in the 3′ → 5′ mRNA decay pathway, following deadenylation and exosome-mediated turnover. The unique structural properties of the cap, which consists of 7-methylguanosine attached to the first transcribed nucleoside by a triphosphate chain (m⁷GpppN), guarantee its resistance to non-specific exonucleases. DcpS enzymes are dimers belonging to the Histidine Triad (HIT) superfamily of pyrophosphatases. The specific hydrolysis of m⁷GpppN by DcpS yields m⁷GMP and NDP. By precluding inhibition of other cap-binding proteins by short m⁷GpppN-containing mRNA fragments, DcpS plays an important role in the cap-dependent mRNA metabolism. Over the past decade, lots of new structural, biochemical and biophysical data on DcpS has accumulated. We attempt to integrate these results, referring to DcpS enzymes from different species. Such a synergistic characteristic of the DcpS structure and activity might be useful for better understanding of the DcpS catalytic mechanism, its regulatory role in gene expression, as well as for designing DcpS inhibitors of potential therapeutic application, e.g. in spinal muscular atrophy.     

The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases

We recently demonstrated that the major decapping activity in mammalian cells involves DcpS, a scavenger pyrophosphatase that hydrolyzes the residual cap structure following 3' to 5' decay of an mRNA. The association of DcpS with 3' to 5' exonuclease exosome components suggests that these two activities are linked and there is a coupled exonucleolytic decay-dependent decapping pathway. We purified DcpS from mammalian cells and identified the cDNA encoding a novel 40 kDa protein possessing DcpS activity. Consistent with purified DcpS, the recombinant protein specifically hydrolyzed methylated cap analog but did not hydrolyze unmethylated cap analog nor did it function on intact capped RNA. Sequence alignments of DcpS from different organisms revealed the presence of a conserved hexapeptide, containing a histidine triad (HIT) sequence with three histidines separated by hydrophobic residues. Mutagenesis analysis revealed that the central histidine within the DcpS HIT motif is critical for decapping activity and defines the HIT motif as a new mRNA decapping domain, making DcpS the first member of the HIT family of proteins with a defined biological function.     

Scavenger decapping activity facilitates 5' to 3' mRNA decay

mRNA degradation occurs through distinct pathways, one primarily from the 5' end of the mRNA and the second from the 3' end. Decay from the 3' end generates the m7GpppN cap dinucleotide, which is subsequently hydrolyzed to m7Gp and ppN in Saccharomyces cerevisiae by a scavenger decapping activity termed Dcs1p. Although Dcs1p functions in the last step of mRNA turnover, we demonstrate that its activity modulates earlier steps of mRNA decay. Disruption of the DCS1 gene manifests a threefold increase of the TIF51A mRNA half-life. Interestingly, the hydrolytic activity of Dcs1p was essential for the altered mRNA turnover, as Dcs1p, but not a catalytically inactive Dcs1p mutant, complemented the increased mRNA stability. Mechanistic analysis revealed that 5' to 3' exoribonucleolytic activity was impeded in the dcs1Delta strain, resulting in the accumulation of uncapped mRNA. These data define a new role for the Dcs1p scavenger decapping enzyme and demonstrate a novel mechanism whereby the final step in the 3' mRNA decay pathway can influence 5' to 3' exoribonucleolytic activity.     

Insights into the structure, mechanism, and regulation of scavenger mRNA decapping activity

Complete removal of residual N-7 guanine cap from degraded messenger RNA is necessary to prevent accumulation of intermediates that might interfere with RNA processing, export, and translation. The human scavenger decapping enzyme, DcpS, catalyzes residual cap hydrolysis following mRNA degradation, releasing N-7 methyl guanosine monophosphate and 5'-diphosphate terminated cap or mRNA products. DcpS structures bound to m(7)GpppG or m(7)GpppA reveal an asymmetric DcpS dimer that simultaneously creates an open nonproductive DcpS-cap complex and a closed productive DcpS-cap complex that alternate via 30 A domain movements. Structural and biochemical analysis suggests an autoregulatory mechanism whereby premature decapping mRNA is prevented by blocking the conformational changes that are required to form a closed productive active site capable of cap hydrolysis.     

Insights into the molecular determinants involved in cap recognition by the vaccinia virus D10 decapping enzyme

Decapping enzymes are required for the removal of the 5′-end ^m7GpppN cap of mRNAs to allow their decay in cells. While many cap-binding proteins recognize the cap structure via the stacking of the methylated guanosine ring between two aromatic residues, the precise mechanism of cap recognition by decapping enzymes has yet to be determined. In order to get insights into the interaction of decapping enzymes with the cap structure, we studied the vaccinia virus D10 decapping enzyme as a model to investigate the important features for substrate recognition by the enzyme. We demonstrate that a number of chemically modified purines can competitively inhibit the decapping reaction, highlighting the molecular features of the cap structure that are required for recognition by the enzyme, such as the nature of the moiety at positions 2 and 6 of the guanine base. A 3D structural model of the D10 protein was generated which suggests amino acids implicated in cap binding. Consequently, we expressed 17 mutant proteins with amino acid substitutions in the active site of D10 and found that eight are critical for the decapping activity. These data underscore the functional features involved in the non-canonical cap-recognition by the vaccinia virus D10 decapping enzyme.     

Components of the Arabidopsis mRNA decapping complex are required for early seedling development

To understand the mechanisms controlling vein patterning in Arabidopsis thaliana, we analyzed two phenotypically similar mutants, varicose (vcs) and trident (tdt). We had previously identified VCS, and recently, human VCS was shown to function in mRNA decapping. Here, we report that TDT encodes the mRNA-decapping enzyme. VCS and TDT function together in small cytoplasmic foci that appear to be processing bodies. To understand the developmental requirements for mRNA decapping, we characterized the vcs and tdt phenotypes. These mutants were small and chlorotic, with severe defects in shoot apical meristem formation and cotyledon vein patterning. Many capped mRNAs accumulated in tdt and vcs mutants, but surprisingly, some mRNAs were specifically depleted. In addition, loss of decapping arrested the decay of some mRNAs, while others showed either modest or no decay defects, suggesting that mRNAs may show specificity for particular decay pathways (3′ to 5′ and 5′ to 3′). Furthermore, the severe block to postembryonic development in vcs and tdt and the accompanying accumulation of embryonic mRNAs indicate that decapping is important for the embryo-to-seedling developmental transition.     

Capped mRNA Degradation Intermediates Accumulate in the Yeast spb8-2 Mutant

mRNA in the yeast Saccharomyces cerevisiae is primarily degraded through a pathway that is stimulated by removal of the mRNA cap structure. Here we report that a mutation in the SPB8 (YJL124c) gene, initially identified as a suppressor mutation of a poly(A)-binding protein (PAB1) gene deletion, stabilizes the mRNA cap structure. Specifically, we find that the spb8-2 mutation results in the accumulation of capped, poly(A)-deficient mRNAs. The presence of this mutation also allows for the detection of mRNA species trimmed from the 3′ end. These data show that this Sm-like protein family member is involved in the process of mRNA decapping, and they provide an example of 3′-5′ mRNA degradation intermediates in yeast.     

miRISC recruits decapping factors to miRNA targets to enhance their degradation

MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5′-to-3′ messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5′ cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.     

mRNA decapping activities and their biological roles

The 5′ cap structure of eukaryotic mRNAs is significant for a variety of cellular events and also serves to protect mRNAs from premature degradation. Analysis of mRNA decay in Saccharomyces cerevisiae has shown that removal of the 5′ cap structure is a key step in the turnover of many yeast mRNAs, and that this decapping is carried out by Dcplp. In addition to the yeast decapping enzyme, other activities that can cleave the 5′ cap structure have been described. These include two mammalian enzymes and two viral activities that cleave cellular mRNA cap structures as part of their life cycle. Here we review these various decapping activities and discuss their biological roles.     

Multiple Nudix family proteins possess mRNA decapping activity

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.     

RNA decapping inside and outside of processing bodies

Decapping is a central step in eukaryotic mRNA turnover. Recent studies have identified several factors involved in catalysis and regulation of decapping. These include the following: an mRNA decapping complex containing the proteins Dcp1 and Dcp2; a nucleolar decapping enzyme, X29, involved in the degradation of U8 snoRNA and perhaps of other capped nuclear RNAs; and a decapping 'scavenger' enzyme, DcpS, that hydrolyzes the cap structure resulting from complete 3'-to-5' degradation of mRNAs by the exosome. Several proteins that stimulate mRNA decapping by the Dcp1:Dcp2 complex co-localize with Dcp1 and Dcp2, together with Xrn1, a 5'-to-3' exonuclease, to structures in the cytoplasm called processing bodies. Recent evidence suggests that the processing bodies may constitute specialized cellular compartments of mRNA turnover, which suggests that mRNA and protein localization may be integral to mRNA decay.     

lsm1 mutations impairing the ability of the Lsm1p-7p-Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo

The poly(A) tail is a crucial determinant in the control of both mRNA translation and decay. Poly(A) tail length dictates the triggering of the degradation of the message body in the major 5′ to 3′ and 3′ to 5′ mRNA decay pathways of eukaryotes. In the 5′ to 3′ pathway oligoadenylated but not polyadenylated mRNAs are selectively decapped in vivo, allowing their subsequent degradation by 5′ to 3′ exonucleolysis. The conserved Lsm1p-7p-Pat1p complex is required for normal rates of decapping in vivo, and the purified complex exhibits strong binding preference for oligoadenylated RNAs over polyadenylated or unadenylated RNAs in vitro. In the present study, we show that two lsm1 mutants produce mutant complexes that fail to exhibit such higher affinity for oligoadenylated RNA in vitro. Interestingly, these mutant complexes are normal with regard to their integrity and retain the characteristic RNA binding properties of the wild-type complex, namely, binding near the 3′-end of the RNA, having higher affinity for unadenylated RNAs that carry U-tracts near the 3′-end over those that do not and exhibiting similar affinities for unadenylated and polyadenylated RNAs. Yet, these lsm1 mutants exhibit a strong mRNA decay defect in vivo. These results underscore the importance of Lsm1p-7p-Pat1p complex–mRNA interaction for mRNA decay in vivo and imply that the oligo(A) tail mediated enhancement of such interaction is crucial in that process.