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.     

Structural and functional control of the eukaryotic mRNA decapping machinery

The regulation of mRNA degradation is critical for proper gene expression. Many major pathways for mRNA decay involve the removal of the 5′ 7-methyl guanosine (m⁷G) cap in the cytoplasm to allow for 5′-to-3′ exonucleolytic decay. The most well studied and conserved eukaryotic decapping enzyme is Dcp2, and its function is aided by co-factors and decapping enhancers. A subset of these factors can act to enhance the catalytic activity of Dcp2, while others might stimulate the remodeling of proteins bound to the mRNA substrate that may otherwise inhibit decapping. Structural studies have provided major insights into the mechanisms by which Dcp2 and decapping co-factors activate decapping. Additional mRNA decay factors can function by recruiting components of the decapping machinery to target mRNAs. mRNA decay factors, decapping factors, and mRNA substrates can be found in cytoplasmic foci named P bodies that are conserved in eukaryotes, though their function remains unknown. In addition to Dcp2, other decapping enzymes have been identified, which may serve to supplement the function of Dcp2 or act in independent decay or quality control pathways. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

The decapping activator Lsm1p-7p-Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs

Decapping is a critical step in mRNA decay. In the 5'-to-3' mRNA decay pathway conserved in all eukaryotes, decay is initiated by poly(A) shortening, and oligoadenylated mRNAs (but not polyadenylated mRNAs) are selectively decapped allowing their subsequent degradation by 5' to 3' exonucleolysis. The highly conserved heptameric Lsm1p-7p complex (made up of the seven Sm-like proteins, Lsm1p-Lsm7p) and its interacting partner Pat1p activate decapping by an unknown mechanism and localize with other decapping factors to the P-bodies in the cytoplasm. The Lsm1p-7p-Pat1p complex also protects the 3'-ends of mRNAs in vivo from trimming, presumably by binding to the 3'-ends. In order to determine the intrinsic RNA-binding properties of this complex, we have purified it from yeast and carried out in vitro analyses. Our studies revealed that it directly binds RNA at/near the 3'-end. Importantly, it possesses the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs such that the former are bound with much higher affinity than the latter. These results indicate that the intrinsic RNA-binding characteristics of this complex form a critical determinant of its in vivo interactions and functions.     

Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs.

The major pathway of eukaryotic mRNA decay involves deadenylation-dependent decapping followed by 5' to 3' exonucleolytic degradation. By examining interactions among mRNA decay factors, the mRNA, and key translation factors, we have identified a critical transition in mRNP organization that leads to decapping and degradation of yeast mRNAs. This transition occurs after deadenylation and includes loss of Pab1p, eIF4E, and eIF4G from the mRNA and association of the decapping activator complex, Lsm1p-7p, which enhances the coimmunoprecipitation of a decapping enzyme complex (Dcp1p and Dcp2p) with the mRNA. These results define an important rearrangement in mRNP organization and suggest that deadenylation promotes mRNA decapping by both the loss of Pab1p and the recruitment of the Lsm1p-7p complex.     

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.     

Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae

The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5'-3' exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5'-3' degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5'-3' exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3Delta had no effect when combined with the lsm1Delta, dhh1Delta, or pat1Delta mutations, which appear to affect an early step in the decapping pathway. This suggests that Edc3p specifically affects the function of the decapping enzyme per se. Consistent with a functional role in decapping, GFP-tagged Edc3p localizes to cytoplasmic foci involved in mRNA decapping referred to as P-bodies. These results identify Edc3p as a new protein involved in the decapping reaction.     

Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection

The decapping of eukaryotic mRNAs is a key step in their degradation. The heteroheptameric Lsm1p-7p complex is a general activator of decapping and also functions in protecting the 3' ends of deadenylated mRNAs from a 3'-trimming reaction. Lsm1p is the unique member of the Lsm1p-7p complex, distinguishing that complex from the functionally different Lsm2p-8p complex. To understand the function of Lsm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNA-binding and inter-subunit interaction residues of Lsm1p led to impairment of mRNA decay, suggesting that the integrity of the Lsm1p-7p complex and the ability of the Lsm1p-7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the Lsm1p-7p complex to the P-bodies; (iii) mRNA 3'-end protection could be indicative of the binding of the Lsm1p-7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3' end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of Lsm1p is also important for mRNA decay function.     

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.     

The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes.

A major pathway of mRNA turnover in eukaryotic cells initiates with deadenylation, leading to mRNA decapping and subsequent 5' to 3' exonuclease digestion. We show that a highly conserved member of the DEAD box family of helicases, Dhh1p, stimulates mRNA decapping in yeast. In dhh1delta mutants, mRNAs accumulate as deadenylated, capped species. Dhh1p's effects on decapping only occur on normal messages as nonsense-mediated decay still occurs in dhh1delta mutants. The role of Dhh1p in decapping appears to be direct, as Dhh1p physically interacts with several proteins involved in mRNA decapping including the decapping enzyme Dcp1p, as well as Lsm1p and Pat1p/Mrt1p, which function to enhance the decapping rate. Additional observations suggest Dhh1p functions to coordinate distinct steps in mRNA function and decay. Dhh1p also associates with Pop2p, a subunit of the mRNA deadenylase. In addition, genetic phenotypes suggest that Dhh1p also has a second biological function. Interestingly, Dhh1p homologs in others species function in maternal mRNA storage. This provides a novel link between the mechanisms of decapping and maternal mRNA translational repression.     

ARE-mRNA degradation requires the 5'-3' decay pathway

As an important mode of suppressing gene expression, messenger RNAs containing an AU-rich element (ARE) in the 3' untranslated region are rapidly degraded in the cytoplasm. ARE-mediated mRNA decay (AMD) is initiated by deadenylation, and in vitro studies have indicated that subsequent degradation occurs in the 3'-5' direction through a complex of exonucleases termed the exosome. An alternative pathway of mRNA degradation occurs at processing bodies, cytoplasmic foci that contain decapping enzymes, the 5'-3' exonuclease Xrn1 and the Lsm1-7 heptamer. To determine which of the two pathways is important for AMD in live cells, we targeted components of both pathways using short interfering RNA in human HT1080 cells. We show that Xrn1 and Lsm1 are essential for AMD. On the other side, out of three exosome components tested, only knockdown of PmScl-75 caused a strong inhibition of AMD. Our results show that mammalian cells, similar to yeast, require the 5'-3' Xrn1 pathway to degrade ARE-mRNAs.     

Localization of AU-rich element-containing mRNA in cytoplasmic granules containing exosome subunits

Eukaryotic mRNAs can be degraded in either decapping/5'-to-3' or 3'-to-5' direction after deadenylation. In yeast and mammalian cells, decay factors involved in the 5'-to-3' decay pathway are concentrated in cytoplasmic processing bodies (P bodies). The mechanistic steps and localization of mammalian mRNA decay are still not completely understood. Here, we investigate functions of human mRNA decay enzymes in AU-rich element (ARE)-mediated mRNA decay (AMD) and find that the deadenylase, poly(A) ribonuclease PARN, and enzymes involved in the 5'-to-3' and 3'-to-5' decay pathways are required for AMD. The ARE-containing reporter mRNA accumulates in discrete cytoplasmic granular structures, which are distinct from P bodies and stress granules. These granules consist of poly(A)-specific ribonuclease, exosome subunits, and decay-promoting ARE-binding proteins. Inhibition of AMD increases accumulation of ARE-mRNA in these granules. We refer to these structures as cytoplasmic exosome granules and suggest that some AMD may occur in these granules.     

The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1

An important control step in the regulation of cytoplasmic mRNA turnover is the removal of the m(7)G cap structure at the 5' end of the message. Here, we describe the functional characterization of Dhh1, a highly conserved member of the family of DEAD box-containing proteins, as a regulator of mRNA decapping in Saccharomyces cerevisiae. Dhh1 is a cytoplasmic protein and is shown to be in a complex with the mRNA degradation factor Pat1/Mtr1 and with the 5'-3' exoribonuclease Xrn1. Dhh1 specifically affects mRNA turnover in the deadenylation-dependent decay pathway, but does not act on the degradation of nonsense-containing mRNAs. Cells that lack dhh1 accumulate degradation intermediates that have lost their poly(A) tail but contain an intact 5' cap structure, suggesting that Dhh1 is required for efficient decapping in vivo. Furthermore, recombinant Dhh1 is able to stimulate the activity of the purified decapping enzyme Dcp1 in an in vitro decapping assay. We propose that the DEAD box protein Dhh1 regulates the access of the decapping enzyme to the m(7)G cap by modulating the structure at the 5' end of mRNAs.