Messenger RNA Turnover in Eukaryotes: Pathways and Enzymes

The control of mRNA degradation is an important component of the regulation of gene expression since the steady-state concentration of mRNA is determined both by the rates of synthesis and of decay. Two general pathways of mRNA decay have been described in eukaryotes. Both pathways share the exonucleolytic removal of the poly(A) tail (deadenylation) as the first step. In one pathway, deadenylation is followed by the hydrolysis of the cap and processive degradation of the mRNA body by a 5′ exonuclease. In the second pathway, the mRNA body is degraded by a complex of 3′ exonucleases before the remaining cap structure is hydrolyzed. This review discusses the proteins involved in the catalysis and control of both decay pathways.     

Degradation of hsp70 and other mRNAs in Drosophila via the 5' 3' pathway and its regulation by heat shock

Two general pathways of mRNA decay have been characterized in yeast. Both start with deadenylation. The major pathway then proceeds via cap hydrolysis and 5'-exonucleolytic degradation whereas the minor pathway consists of 3'-exonucleolytic decay followed by hydrolysis of the remaining cap structure. In higher eukaryotes, these pathways of mRNA decay are believed to be conserved but have not been well characterized. We have investigated the decay of the hsp70 mRNA in Drosophila Schneider cells. As shown by the use of reporter constructs, rapid deadenylation of this mRNA is directed by its 3'-untranslated region. The main deadenylase is the CCR4.NOT complex; the PAN nuclease makes a lesser contribution. Heat shock prevents deadenylation not only of the hsp70 but also of bulk mRNA. A completely deadenylated capped hsp70 mRNA decay intermediate accumulates transiently and is degraded via cap hydrolysis and 5'-decay. Thus, decapping is a slow step in the degradation pathway. Cap hydrolysis is also inhibited during heat shock. Degradation of reporter RNAs from the 3'-end became detectable only upon inhibition of 5'-decay and thus represents a minor decay pathway. Because two reporter RNAs and at least two endogenous mRNAs were degraded primarily from the 5'-end with cap hydrolysis as a slow step, this pathway appears to be of general importance for mRNA decay in Drosophila.     

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5'-to-3' degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.     

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.     

Messenger RNA turnover in eukaryotes: pathways and enzymes

The control of mRNA degradation is an important component of the regulation of gene expression since the steady-state concentration of mRNA is determined both by the rates of synthesis and of decay. Two general pathways of mRNA decay have been described in eukaryotes. Both pathways share the exonucleolytic removal of the poly(A) tail (deadenylation) as the first step. In one pathway, deadenylation is followed by the hydrolysis of the cap and processive degradation of the mRNA body by a 5' exonuclease. In the second pathway, the mRNA body is degraded by a complex of 3' exonucleases before the remaining cap structure is hydrolyzed. This review discusses the proteins involved in the catalysis and control of both decay pathways.     

LSM1 over-expression in Saccharomyces cerevisiae depletes U6 snRNA levels

Lsm1 is a component of the Lsm1-7 complex involved in cytoplasmic mRNA degradation. Lsm1 is over-expressed in multiple tumor types, including over 80% of pancreatic tumors, and increased levels of Lsm1 protein have been shown to induce carcinogenic effects. Therefore, understanding the perturbations in cell process due to increased Lsm1 protein may help to identify possible therapeutics targeting tumors over-expressing Lsm1. Herein, we show that LSM1 over-expression in the yeast Saccharomyces cerevisiae inhibits growth primarily due to U6 snRNA depletion, thereby altering pre-mRNA splicing. The decrease in U6 snRNA levels causes yeast strains over-expressing Lsm1 to be hypersensitive to loss of other proteins required for production or function of the U6 snRNA, supporting a model wherein excess Lsm1 reduces the availability of the Lsm2-7 proteins, which also assemble with Lsm8 to form a complex that binds and stabilizes the U6 snRNA. Yeast strains over-expressing Lsm1 also display minor alterations in mRNA decay and demonstrate increased susceptibility to mutations inhibiting cytoplasmic deadenylation, a process required for both 5′-to-3′ and 3′-to-5′ pathways of exonucleolytic decay. These results suggest that inhibition of splicing and/or deadenylation may be effective therapies for Lsm1-over-expressing tumors.     

The mammalian exosome mediates the efficient degradation of mRNAs that contain AU-rich elements.

HeLa cytoplasmic extracts contain both 3'-5' and 5'-3' exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate-modified RNAs. These data indicate that 3'-5' exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immunodepletion using antibodies specific for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for efficient 3'-5' exonucleolytic decay. Furthermore, 3'-5' exonucleolytic decay was stimulated dramatically by AU-rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM-Scl75 protein was found to interact specifically with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the efficiency of ARE-containing mRNA turnover.     

Poly(A)-binding proteins regulate both mRNA deadenylation and decapping in yeast cytoplasmic extracts.

The pathway of mRNA degradation has been extensively studied in the yeast, Saccharomyces cerevisiae, and it is now clear that many mRNAs decay by a deadenylation-dependent mechanism. Although several of the factors required for mRNA decay have been identified, the regulation and precise roles of many of the proteins involved remains unclear. We have developed an in vitro system that recapitulates both the deadenylation and the decapping steps of mRNA decay. Furthermore, both deadenylation and decapping are inhibited by poly(A) binding proteins in our assay. Our system has allowed us to separate the decay process from translation and we have shown that the poly(A) tail is capable of inhibiting decapping in an eIF4E-independent manner. Our in vitro system should prove invaluable in dissecting the mechanisms of mRNA turnover.     

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.     

A cell-free mRNA stability assay reveals conservation of the enzymes and mechanisms of mRNA decay between mosquito and mammalian cell lines

The rate of mRNA turnover is an important determinant of levels of gene expression. Although this process has been studied extensively in mammalian cells and yeast, relatively little is known about the mRNA decay pathways in insects. Our analysis found that the vast majority of components of the mRNA decay machinery are conserved between humans and mosquitoes. Moreover, the half-lives of Aedes albopictus mRNAs are within a similar range to those of mammalian mRNAs. In order to investigate mechanistic aspects of mRNA decay in mosquitoes, we developed an in vitro system using cytoplasmic S100 extracts from A. albopictus C6/36 cells. Using this decay assay, we show here that all the pathways of mRNA turnover that have been observed in mammalian cells (deadenylation, decapping, 3′-to-5′ exonucleolytic decay and 5′-to-3′ exonucleolytic decay) are active in C6/36 extracts. Finally, we present compelling evidence that the major deadenylase in C6/36 extracts is likely to be a homolog of the human poly(A) specific ribonuclease, PARN. Our results suggest a high level of conservation in the factors and pathways of mRNA decay between mosquitoes and humans.     

dTIS11 Protein-dependent Polysomal Deadenylation Is the Key Step in AU-rich Element-mediated mRNA Decay in Drosophila Cells

The destabilization of AU-rich element (ARE)-containing mRNAs mediated by proteins of the TIS11 family is conserved among eukaryotes including Drosophila. Previous studies have demonstrated that Tristetraprolin, a human protein of the TIS11 family, induces the degradation of ARE-containing mRNAs through a large variety of mechanisms including deadenylation, decapping, and P-body targeting. We have previously shown that the degradation of the mRNA encoding the antimicrobial peptide Cecropin A1 (CecA1) is controlled by the TIS11 protein (dTIS11) in Drosophila cells. In this study, we used CecA1 mRNA as a model to investigate the molecular mechanism of dTIS11-mediated mRNA decay. We observed that during the biphasic deadenylation and decay process of this mRNA, dTIS11 enhances deadenylation performed by the CCR4-CAF-NOT complex while the mRNA is still associated with ribosomes. Sequencing of mRNA degradation intermediates revealed that the complete deadenylation of the mRNA triggers its decapping and decay in both the 5′-3′ and the 3′-5′ directions. Contrary to the observations made for its mammalian homologs, overexpression of dTIS11 does not promote the localization of ARE-containing mRNAs in P-bodies but rather decreases the accumulation of CecA1 mRNA in these structures by enhancing the degradation process. Therefore, our results suggest that proteins of the TIS11 family may have acquired additional functions in the course of evolution from invertebrates to mammals.