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The Interplay between Eukaryotic mRNA Degradation and Translation
The Interplay between Eukaryotic mRNA Degradation and Translation W Hu, Whitehead Institute for Biomedical Research, Cambridge, MA, USA r 2016 Elsevier Inc. All rights reserved.
The destruction of mRNA is essential for proper regulation of gene expression. All mRNA must be destroyed to ensure that nuclear regulatory decisions are manifested as cytoplasmic events. In other words, ceasing mRNA transcription in response to environmental or physiological cues is irrelevant as long as cytoplasmic mRNA exists and is translated. From this, it can be said that mRNA decay contributes significantly to the proteomes’ overall architecture by allowing cells to quickly adapt to environmental and physiological changes. Although much is known about mRNA decay in eukaryotes, understanding how the process is modulated is far from complete. In this section, we first discuss our current understanding of eukaryotic mRNA decay. Then we put mRNA degradation in the context of mRNA translation, which is another important cyctoplasmic event of gene expression, as these two events are known to be tightly integrated with each other for decades. In the last few years, many studies have concentrated on this relationship as a means to elucidate regulation of mRNA degradation. From this, two views have arisen. The first suggests that mRNA must be removed from ribosomes to be destroyed in ribosome-free areas, such as P-bodies. The other view proposes mRNA decay occurs in concert with protein synthesis. Here we summarize our current understanding of how eukaryotic mRNA decay interconnects with mRNA translation, and then analyze the data that have lead to these two diametrically opposed models of where mRNA decay occurs within the cell. Finally, we attempt to reconcile these two views and suggest important areas for future investigation.
Overview of Eukaryotic mRNA turnover Cytoplasmic mRNAs are destroyed by two major pathways. Both are initiated by the exonucleolytic digestion of the 3′ poly (A) tail (a process termed deadenylation). Deadenylation is a key regulatory step in mRNA half-life determination because its rate is highly variable among transcripts. In some cases, deadenylation leads to mRNA destruction 3′–5′ by the cytoplasmic exosome. In other cases, deadenylation causes the rapid removal of the 5′ 7mGpppN cap and digestion of the transcript body 5′–3′ (reviewed in Coller and Parker, 2004). Which pathway predominates is not clearly understood. Most likely transcript, tissue, and species specific differences dictate the predominate pathway employed. In this article, we focus exclusively on the deadenylation-dependent decapping pathway since it has been extensively characterized and its relationship to translation is more established than that of the cytoplasmic exosome-dependent pathway. As mentioned, deadenylation is the first step of mRNA decay and is often thought to be rate limiting. This reaction is carried out by several enzymes (deadenylases) that digest the poly(A) tail in a 3′–5′ direction. Generally, mRNA deadenylation is biphasic, the first step is initiated by the deadenylase dependent
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poly(A) nuclease 2 (PAN2). Following this initial trimming event, the bulk of the tail is digested by the enzyme complex CCR4/POP2/NOT (Yamashita et al., 2005). Other deadenylases, however, exist including poly(A)-specific ribonuclease (PARN), Nocturin, and Angel proteins (reviewed in Goldstrohm and Wickens, 2008). It would appear, then, that deadenylation is facilitated by a seemingly redundant class of enzymes. Diversity in transcript stability might occur in part by the manner in which these enzymes achieve transcript specificity. For example, 3′UTR-binding proteins and miRNAs can enhance deadenylation by tethering a specific enzyme (Giraldez et al., 2006). In yeast as well as in mammalian cells, deadenylation usually leads to destruction of the mRNA. In some systems, deadenylation can lead to translational storage of the mRNA. Although still unclear, it is thought that the poly(A) tail mediates its effect on mRNA translation and decay by way of an interaction between the poly(A)-binding protein (PAB) and the translation initiation factor eIF4G (Coller et al., 1998; Jacobson and Peltz, 1996). Deadenylation serves to displace PAB from the mRNA thereby liberating its protective and/or translational stimulatory function. In the cases where transcript destruction follows deadenylation, once the mRNA tail has been trimmed to a length no longer supporting PAB binding, the 7-methyl-guanosine cap structure is rapidly cleaved from the mRNA’s 5′ end. The decapping reaction requires the enzymatic activity of the DCP2 protein. Enzymatically, the pyrophosphatase activity of DCP2 cleaves the alpha-beta bond of the 5′ cap liberating m7′GDP and leaving a 5′ monophosphate on the transcript. In vitro, DCP2 is sufficient for this activity (Lykke-Andersen, 2002). Although simple in chemistry in vitro, the in vivo orchestration of mRNA decapping requires a suite of proteins including DCP1, DHH1(RCK/p54), Hedls (only in metazoans), Pat1p (P100), Lsm1–7, Lsm12, Scd6 (Trailerhitch...), and Edc1–3 (reviewed in Franks and Lykke-Andersen, 2008). The precise function(s) of these factors is murky. Some decapping activators like LSM1–7 and Hedls may serve structural roles in promoting binding of DCP2 to the mRNA. Others like DHH1 and PAT1 are hypothesized to promote DCP2’s accessibility to the cap by dissociating factors bound at the mRNA 5′ end, such as eIF4F (Coller and Parker, 2005; Chu and Rana, 2006). Irrespective of their true function, the complexity of this process indicates that mRNA decapping is tightly regulated. Control of decapping makes sense since this step of decay commits the mRNA to destruction and unlike deadenylation is largely irreversible although exceptions have been documented (reviewed in Schoenberg and Maquat, 2009). Subsequent to decapping, the transcript is quickly and efficiently destroyed by the 5′–3′ exonuclease, XRN1. This enzyme is highly processive and decay intermediates are hard to detect (Stevens, 2001). To date, no regulation of XRN1 activity has been detected, thus, it is generally thought that the 5′–3′ exonucleolytic decay is not a rate-limiting step in turnover.
Encyclopedia of Cell Biology, Volume 1
doi:10.1016/B978-0-12-394447-4.10057-4
The Interplay between Eukaryotic mRNA Degradation and Translation
mRNA Degradation is Intimately Linked to Translation All mRNA must and will succumb to decay. Transcript decay is, therefore, a default state. The spectrum of observed half-lives most likely represents acceleration or deceleration of this default rate. The translatability of an mRNA is a critical contributor to its overall stability (reviewed in Franks and Lykke-Andersen, 2008; Jacobson and Peltz, 1996). Specifically, an inverse correlation has been established showing efficiently translated mRNAs have longer half-lives; while poorly translated mRNAs are unstable. These findings suggest that a major determinant of mRNA stability is the translation machinery. Curiously, however, modulation of each specific sub-step of translation, i.e., initiation, elongation, or termination, is perceived to have disparate effects on mRNA’s half-life. It is important to note that this perception is based largely on the manner of analysis (see below). The when and where of mRNA decapping and exonucleolytic decay has been the subject of some debate. Models for where mRNA decay takes place are in part based on the distinct manner in which translational inhibition affects mRNA decay. Experimental evidence has suggested two possibilities. On one side data seems to indicate that before decapping can occur, the translational machinery must be liberated and then the message is destroyed in specialized places within the cell. On the other side, there are data to suggest that mRNA never leave polysomes and are degraded co-translationally. In the following sections we highlight the work of many individuals whose observations have generated these two distinct views.
The Ying-Yang of Translational Initiation and Transcript Stability Translational initiation is posited to be in direct competition with mRNA decapping (reviewed in Franks and Lykke-Andersen, 2008). From a priori view, this hypothesis makes sense because both translational initiation and mRNA decapping are dependent on factors binding on or near the 5′ cap (Cougot et al., 2004). This theory is also supported by experimental data. In a simplified view, translation initiation consists of three major events: first recognition of the cap by eIF4F, which is composed of eIF4E, eIF4G, and eIF4A; second, recruitment of the 40 S ribosomal subunit; third, scanning of the 40 S ribosomal subunit to the first AUG (reviewed in Kapp and Lorsch, 2004). Altering the rate of any of these three events has a profound effect on mRNA half-live. For instance, mutations in eIF4F reducing its affinity for the cap result in rapid and efficient deadenylation and decapping (Schwartz and Parker, 1999). Similarly, inhibiting 40 S ribosomal subunit recruitment and scanning accelerates mRNA deadenylation and decapping. For example, inactivation of eIF3, thereby blocking 40 S recruitment (Kapp and Lorsch, 2004), results in rapid mRNA decay (Heikkinen et al., 2003). Cis-acting RNA structures that inhibit 40 S ribosomal subunit scanning also accelerate mRNA turnover (Muhlrad et al., 1995; Coller and Parker, 2005). Lastly, the context of the AUG start codon influences mRNA stability. Specifically, nucleotide changes around the translation start codon predicted to decrease translational efficiency also dramatically destabilize reporter transcripts (LaGrandeur and
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Parker, 1999). Taken together these correlations between translational initiation and mRNA decapping predict that decreasing ribosome occupancy on the 5′UTR allows the decay machinery to associate more efficiently with the transcript. Moreover, these data suggest that the translation initiation factors and decapping factors vie for the mRNA in vivo. A direct competition between decapping and eIF4E has been documented in vitro. Purification of DCP1 from yeast extracts allows decapping to be monitored in vitro (presumable because purification of DCP1 brings along the decapping enzyme DCP2). Addition of recombinant eIF4E protein efficiently inhibits in vitro decapping mediated by the purified decapping enzyme (Schwartz and Parker, 2000). In conjunction with the aforementioned in vivo work, these data have led to the model that dissociation of eIF4E is required before mRNA decapping can occur. Importantly, this notion is supported by genetic analysis. Specifically, loss of eIF4E will restore the decapping activity when it is partially impaired genetically (i.e., DCP1Ts; Schwartz and Parker, 2000). Collectively, these results indicate that the mRNA decapping enzyme (Dcp2/Dcp1) and eIF4E are probably in competition for the 5′-end cap structure on an mRNA. Clearly, mRNA decapping is stimulated by loss of eIF4E function, but it is also important to note that in vivo inhibition of translational initiation in any manner also greatly enhances deadenylation rate. The effects of translation rate on deadenylation have remained unexplored. Undoubtedly there is a positive correlation between the efficiency of translational initiation and mRNA stability. The displacement of eIF4E from the cap, therefore, is proposed to be a necessary first step in mRNA decapping (reviewed in Coller and Parker, 2004). At this point, it is important to note that the inverse correlation between translation rate and mRNA decapping does not hold true under every physiological condition. For example, cellular stress can have profound effects on translational initiation and mRNA decay. Under these extreme conditions the predictable correlation between translation and decay does not occur. Specifically, translational initiation is inhibited when cells are deprived of glucose (Ashe et al., 2000). Despite this, mRNAs are dramatically stabilized rather than destabilized (Hilgers et al., 2006). On the other hand, amino acid deprivation also powerfully inhibits translation initiation (ref) but mRNAs are greatly destabilized under this condition. Lastly, the unfolded protein response also triggers repression of translation (Scheuner et al., 2001), but this stress does not affect mRNA stability at all (Hilgers et al., 2006). One possible explanation for these discrepancies is that each distinct stress alters one or more of the mRNA decay factors. Alternatively, each stress might change mRNP dynamics and therefore the manner by which it is perceived by the decay machinery. It seems likely, therefore, that a more detailed understanding of how cellular stress impacts mRNA stability will shed more light on the complex interplay between decay and translation.
Translation Elongation and mRNA Turnover It is postulated that once an mRNA is engaged in elongation, ribosomes provide a protective quality that insulates the
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transcript from decay (Jacobson and Peltz, 1996). This theory, however, is controversial. Unlike translational initiation where slowing the process enhances decay, the manner in which elongation is perturbed dramatically influences the result obtained. For example, treating cells with the translation elongation inhibitor, cycloheximide (a drug that freezes ribosomes on mRNA), results in a significant inhibition of decay in budding yeast and high eukaryotes (Beelman and Parker, 1994; Ross, 1995). Similar results are obtained when elongation is stopped using genetic mutation. Specifically, depleting charged tRNAs by inhibiting maturation of their 3′ end will dramatically stabilize mRNA (Peltz et al., 1992). In both cases, blocking elongation is suggested to inhibit decapping (Beelman and Parker, 1994), deadenylation still occurs, albeit at a slightly reduced rate (Hilgers et al., 2006). It is important to point out, that both of these procedures do not kinetically slow elongation, rather, they freeze ribosomes on mRNA and block elongation altogether. Paradoxically, slowing the rate of translation elongation is similar to slowing translational initiation in that it destabilizes mRNAs. For example, rare codons are recognized by tRNAs with low concentrations within the cell, thus, mRNAs with rare codons have a relatively slow translation rate compared to the mRNAs with cognate normal codons. The presence of rare codons within an mRNA saturates the transcript with ribosomes and dramatically shortens halflife (Caponigro et al., 1993; Hu et al., 2009). Slowing ribosome translocation in this manner, therefore, destabilizes mRNAs. One possible explanation for these differences is that freezing mRNA on ribosomes may ‘lock’ the transcript in an RNP conformation that is resistant to mRNA decay factors. Conversely slowing elongation enhances decay by a yet unknown mechanism.
Translation Termination and mRNA Stability Eukaryotic mRNA translation termination requires two release factors, eRF1 and eRF3. Translation termination process can influence mRNA half-life. Specifically, it was observed that the N-terminal domain of eRF3, which is not required for translation termination, can interact with Pab1, and this interaction is involved in modulating mRNA stability (Hosoda et al., 2003). Disruption of this interaction results in translationdependent stabilization of mRNA caused by decreased deadenylation rate (Hosoda et al., 2003). Interestingly, it was further found that certain deadenylase complexes can also bind to the same site on Pab1 that is involved in the interaction with eRF3 (Funakoshi et al., 2007). Thus, it has been postulated that eRF3 can regulate mRNA deadenylation by competitively binding to the Pab1, which then modulates the recruitment and activation of deadenylase complexes (Funakoshi et al., 2007). In addition to the release factors, other proteins that can modulate translation termination can also influence mRNA stability. For example, a recent characterized protein named Tpa1 can interact with the two release factors and regulate the readthrough of stop codons (Keeling et al., 2006). Interestingly, although the detailed mechanisms still remain elusive, knocking out this protein can have decreased deadenylation rate and increased mRNA stability (Keeling et al., 2006). Collectively, these results suggest that
mRNA translation termination can results in mRNP conformational changes that can influence mRNA stability, likely via modulating mRNA deadenylation.
Where Does Eukaryotic mRNA Degradation Occur Within the Cell? In recent years, understanding where mRNA degradation occurs within the cell has been given a great deal of scientific investigation. The thought is that insight into how the large repertoire of observed mRNA half-lives is achieved may come from knowing when and where degradation occurs within the cytoplasm. At present, there are two different views on where mRNA degradation takes place. The first comes from the discovery that mRNA decapping and decay factors aggregate into punctuate, microscopically visible structures. These structures have been given the epitaph of processing bodies or P-bodies for short. Importantly, P-bodies do not contain ribosomes nor do they contain most translational initiation factors. What they do contain is the full complement of decapping proteins, certain mRNA decay intermediates, and in higher eukaryotes, the miRNA machinery (reviewed in Franks and Lykke-Andersen, 2008; Parker and Sheth, 2007). In conjunction with the aforementioned findings that mRNA translation is generally positively correlated with half-life, these data have led to a popular ‘two-step’ model for regulating transcript stability in which ribosome dissociation first occurs, then the mRNA is trafficked to P-bodies where mRNA decapping ensues (reviewed in Franks and Lykke-Andersen, 2008; Parker and Sheth, 2007). We refer to this model as the ‘mRNA cycle hypothesis,’ because it is postulated that mRNA can cycle in and out of polyribosomes and into a quiescent state that can be either stored/reutilized or degraded (Brengues et al., 2005). A second model supported in the literature, is that mRNA decay does not involve a fundamental transition between a polyribosomebound state and a translationally repressed state, but rather occurs co-translationally (Hu et al., 2009). This hypothesis dates back almost 20 years (Beelman and Parker, 1994; Mangus and Jacobson, 1999), but has lacked strong experimental evidence until recently. Nonetheless, both models are supported but have their limitations. Here, we will examine these two models in detail and at the end attempt to provide a unifying theory.
The mRNA Cycle Hypothesis – From Polysome to p-Bodies (and Perhaps Back) The theory that translation and decay are partitioned events is based in part on the aforementioned distinct effects translation initiation versus elongation have on mRNA half-life. The mRNA cycle hypothesis suggests that mRNA is translated and then at some point becomes translationally silenced and moved into a state of quiescence. Once this inactive state is achieved, the mRNA can either be destroyed or perhaps reutilized in response to certain cues. In theory, translational repression of mRNA makes sense from a logistical stand point. The 5′-cap structure is occupied by a suite of proteins that promote 40 S ribosome joining. At some
The Interplay between Eukaryotic mRNA Degradation and Translation
point, DCP2 must have access to the cap and 5′UTR, and thus it seems logical that the 5′ end is remodeled and initiation factors displaced (Steiger et al., 2003). It is predicted that the downstream consequence of this remodeling must be translational repression, as manifest by ribosome dissociation. The RNA cycle hypothesis is rooted in studies of translational initiation and its effect on decay. As discussed in the previous section, Schwartz and Parker (1999) found that mutations in eIF4F result in mRNA destabilization. Cap binding by eIF4F, therefore was proposed to be in competition with mRNA decapping. Similar results were seen when other aspects of initiation were compromised, including 40 S joining and AUG recognition. Moreover, in vitro experiments demonstrated that addition of purified eIF-4E could reduce decapping in cell extracts (Schwartz and Parker, 2000). The combined findings were interpreted as a direct competition between eIF4E and DCP2 exists. A corollary of this hypothesis is that translational repression, defined as ribosome dissociation, is required before mRNA decapping. The mRNA cycle model is also supported by the discovery that several decapping regulators influence mRNA translation. Specifically, DHH1 and PAT1 were shown to be bona fide translational repressors in yeast and humans (Coller and Parker, 2005; Chu and Rana, 2006). Consistent with the notion, DHH1 is homologous to factors implicated in maternal mRNA storage (reviewed in Rajyaguru and Parker, 2009) thus decapping was hypothesized to be similar to posttranscriptional events that occur during early development (Coller et al., 2001). The nature of how mRNA decapping regulators influence mRNA translation is still unknown, but has been proposed to inhibit translation initiation (Coller and Parker, 2005). Other aspects of mRNA decay are also thought to result in translational silencing, and possible ribosome dissociation. Specifically, mRNA deadenylation can lead to translational repression (Huarte et al., 1992). This is especially well documented during development. The poly(A) tail and its binding protein, PAB, are hypothesized to interact with the 5′ cap via a protein interaction with eIF4G. Poly(A) is popularly believed to stimulate translation by way of this ‘close-loop’ (Jacobson and Peltz, 1996). Collectively, the first step of mRNA decay, deadenylation, would be predicted to result in a loss of translational efficiency. It is important to note, however, that controversy exists whether deadenylation is a cause or effect of translational repression in some context. P-bodies, above all things, bolstered the notion that ribosome dissociation is required for mRNA decapping and that these events are similar to storage events that occur in other contexts (reviewed in Parker and Sheth, 2007; Rajyaguru and Parker, 2009). The discovery of P-bodies dates back to 1997 when Bashkirov et al. (1997) cloned the mouse homolog of the known 5′–3′ exonuclease Xrn1. Using mouse E10 fibroblast cells, it was observed that mXrn1 localized to punctuate cytoplasmic structures. At this time, the authors proposed that these structures may represent either sites for RNA turnover or sites in which the enzyme is stored until used. P-bodies were rediscovered in 2002 when Eystathioy et al. (2002) found that the human auto-antigen protein, GW182, was also found in cytoplasmic granules. Although it was not clear at this time,
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GW182 is a critical factor in mediating miRNA translational control (Tritschler et al., 2010). One year later, a flurry of studies simultaneously demonstrated that, in the addition to XRN1, the major decapping factors also are found in cytoplasmic foci (many refs). Sheth and Parker (2003) extended this analysis by showing that mRNA decay intermediates co-localized with these granules in a manner that is dependent on the enzymatic activity of yeast XRN1. Similar results were seen in humans (Cougot et al., 2004). Thus the aggregation of decay factors in granules was suggested to be of functional significance and that these foci represented the place where mRNAs were degraded. P-bodies are devoid of ribosomes and other vital translational initiation factors (Sheth and Parker, 2003; Cougot et al., 2004). Coupled with the aforementioned results indicating decapping is in competition with translational initiation, it was further proposed that mRNA must be removed from ribosomes and trafficked to a P-body before decay ensues (reviewed in Franks and Lykke-Andersen, 2008). Indeed the very term P-bodies, or processing bodies was coined, in part, to draw similarity to P-granules or polar bodies, known sites of mRNA storage in germ-lines, oocytes, and embryos (reviewed in Parker and Sheth, 2007; Rajyaguru and Parker, 2009). Consistent with this, P-bodies have also been suggested to contain translational quiescent mRNA (Brengues et al., 2005; Parker and Sheth, 2007; Franks and LykkeAndersen, 2008). In mammalian systems, the miRNA machinery co-localizes to P-bodies further supporting a model in which P-bodies are sites of mRNA storage. In yeast, stress conditions like glucose deprivation result in global shut-down of mRNA translation and mRNA decay. This correlates with an increase in P-body size and abundance. An increase in P-body size has been interpreted as an influx of mRNA into these structures (Brengues et al., 2005; Sheth and Parker, 2003). Taken together, mRNA decapping has hypothesized to require removal of mRNA from the translational apparatus followed by packaging into P-bodies where either decay or storage ensues. The RNA cycle model is very provocative, but there are still many questions left to be answered. For instance, one important feature of this hypothesis is that DCP2 and eIF-4E are in direct competition with each other for cap access. While this competition is clear in vitro, in vivo the relationship between DCP2 and eIF4E is more complex. Specifically, if a simple competitive relationship existed, then loss of eIF4E function would result in promiscuous mRNA decapping independent of deadenylation. Although perturbations in initiation result in accelerated decapping, in all cases when initiation is impaired (i.e., initiation factor mutants, AUG context changes, and cis-acting initiation blocks) decapping is not uncoupled from deadenylation. Indeed, loss of deadenylation by ccr4 mutation stabilizes non-translating mRNAs. Rather, loss of eIF-4F function results in rapid deadenylation followed by rapid decapping (Schwartz and Parker, 1999). These data indicate that loss of eIF-4E is not sufficient to elicit decapping directly, rather dissolution of the initiation complex sensitizes the mRNA toward decay in a fashion that is still unknown. In this light, the notion that eIF-4E displacement is a critical event in decapping stimulated by deadenylation requires reinvestigation.
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The RNA cycle hypothesis is also based on the notion that removing mRNAs from ribosomes accelerates their decay. Based on this, polysomes would provide a protective quality to the transcript, for example, sequestering the message from P-bodies. While generally, impeding translational initiation promotes rapid mRNA decay, important caveats exist. For instance, there is no correlation between changes of translation status and mRNA stability under conditions of stress known to impede protein synthesis (see above). Moreover, little evidence exists that ribosomes protect the mRNA from destruction. In fact, slowing translation elongation by use of rare codons saturates the message with ribosomes, but, paradoxically, greatly enhances its decay by the normal deadenylation-dependent decapping machinery. It appears then that an mRNA that never has ribosomes is degraded quickly, and an mRNA that is saturated with slowly moving ribosomes is also degraded quickly. Thus, there is no obvious need for the mRNA to dissociate from the translational machinery in order to be degraded efficiently (see below). Lastly, an important aspect of the RNA cycle hypothesis is that once mRNA leaves ribosomes they enter P-bodies. From a quantitative point of view, it is still unclear to what extend mRNA decapping and 5′–3′ exonucleolytic decay occur in ribosome-free areas in the cell. Moreover, it is unclear how much of the decapping complex assembles into P-bodies versus the soluble cytosol. In some cases, it has been suggested to be only a minor fraction. For instance quantification of the P-body protein Ago2 in mammalian cells revealed that less than 5% of total Ago2 is localized in P-bodies (Leung et al., 2006). Second, P-bodies, whose abundance correlates well with events that enhance translation repression and mRNA decay, are not required for either of these events. Specifically, in both yeast and higher eukaryotes, several groups have shown that P-body formation can be uncoupled from mRNA turnover and translation repression (Sweet et al., 2007; Decker et al., 2007; Eulalio et al., 2007). Together, these data demonstrate that the function of P-bodies remains enigmatic. Clearly, however, the aggregation of decapping factors into P-bodies is not required for normal rates of mRNA turnover. In summary, the RNA cycle hypothesis is provocative. It provides a simple explanation for how mRNA turnover and translation are interconnected. Importantly, however, significant caveats exist suggesting that mRNA turnover is not as simplistic as the model suggest.
Parting on Polysomes A second model that has emerged in the literature is that message decay takes place co-translationally. In other words, mRNA decapping and 5′–3′ exonucleolytic digestion occur primarily on polyribosomes and not in a ribosome-free state (e.g., a P-body). The hypothesis that mRNAs are destroyed on polysomes is not new. In most cases, however, this scenario has only been observed in special cases; specifically albumin and β-tubulin mRNA (Pastori et al., 1991; Theodorakis and Cleveland, 1992). Nonetheless, small clues are seen throughout the literature. Early evidence was first seen yeast. As mentioned in previous sections, treating yeast cells with the translational elongation inhibitor cycloheximide leads to the
dramatic stabilization of mRNAs. Recent interpretations of this effect are that the mRNA is sequestered in polyribosomes under these conditions and away from P-bodies (reviewed in Parker and Sheth, 2007; Franks and Lykke-Andersen, 2008). Interestingly, however, Beelman and Parker (1994) investigated mRNA decay in cells treated with cycloheximide and found the mRNA accumulated as a slightly shorter species over time. Although this finding wasn’t further characterized, they proposed the truncated mRNA was the result of decapping and digestion up to a ribosome stalled at the AUG. Consistent with the hypothesis of co-translational decapping/decay, it was also found that in humans decapping activity co-sediments with polysomes (Wang et al., 2002a,b). Mangus and Jacobson (1999) observed that decay intermediates similar to those observed in P-bodies were also associated with polyribosomes. Lastly, we provided additional supportive by showing that the majority of decapping is observed while the transcript is saturated with ribosomes (Hu et al., 2009). Collectively, these data argue that under normal physiological conditions, mRNA decapping and 5′–3′ exonucleolytic digestion occur on polyribosomes. A corollary of this hypothesis is that a transition into a translational repressed state, a predicted by the RNA cycle hypothesis, is not required for efficient mRNA decay. Like the RNA cycle hypothesis, the model of co-translational decay also has important caveats to mention. First, numerous reports have shown that events like deadenylation inhibit protein synthesis (Huarte et al., 1992; Thompson et al., 2000; Beilharz et al., 2009), yet no apparent difference in ribosome association can be seen for mRNA before and after deadenylation in our analysis. It is important to note here that polysome analysis provides direct physical evidence for the binding of ribosomes to an mRNA, it does not provide any information about translation rate. For example, it has been documented that miRNA targets are saturated with ribosomes, yet no protein output is detectable (Maroney et al., 2006; Nottrott et al., 2006). It is unclear how this is possible, but most likely indicates that there are enigmatic mechanisms of translational control that have yet to be revealed. Indeed in our own analysis we cannot conclude if nonadenylated mRNAs are translating at the same rate as fully adenylated mRNA. A clear and important area of future investigation is to correlate ribosome occupancy with adenylation status and protein output. Perhaps this analysis will shed light on this unexpected finding. Second, it is clear that the decapping regulators DHH1 and PAT1 inhibit protein synthesis most likely by altering translational initiation (Coller and Parker, 2005) and that mutations in initiation factors can accelerate decapping. In this regard, it is important to realize that polysome analysis is a gross measurement of ribosome-associated mRNPs. Subtle changes such as rearrangement of the mRNA's 5′ UTR are beyond the detection limit of this assay, unless they result in dramatic changes in polysome association. Lastly, the contribution of initiation factors to polyribosome association/maintenance is still unclear. In recent years it has been observed that loss of initiation factor function in vivo has only a mild effect on polyribosome formation. Thus, it is formally possible that 5′UTR remodeling can occur with little or no appreciable change in ribosome occupancy. The explanation for this paradox remains unclear, but might indicate that initiation factors are only rate limiting for
The Interplay between Eukaryotic mRNA Degradation and Translation
translation under certain circumstances or during early events in protein synthesis before the establishment of polyribosomes.
What Does It All Mean? The interplay between translation and message decay is of great importance for understanding the overall regulation of gene expression. As discussed in the previous two sections, there are two points of view. One is that most mRNA decay occurs after ribosome dissociation and perhaps in P-bodies. The other suggests that mRNA never leave ribosomes and decay is co-translational. Both hypothesizes are based on clear experimental observations. Nether model is perfect and both have important caveats for consideration. In the end, the truth most likely lies somewhere in between. Biochemical data indicate that polyribosomes represent a major site of mRNA decapping within the cell under normal growth conditions. Importantly, however, evidence also exists to suggest that decay occurs, at some level, in P-bodies. We propose that all mRNA degradation is initiated on polyribosomes at the step of deadenylation. Where subsequent transcript degradation occurs is a function of the messages’ translation rate versus relative to its decapping rate. We hypothesize that deadenylation does indeed reduces translational efficiency (Huarte et al., 1992) perhaps through loss of the PAB, Pab1p (Jacobson and Peltz, 1996), or association of decapping regulators such as DHH1 and PAT1 (Coller and Parker, 2005). These events allow for the remodeling of the 5′UTR and deposition of a decapping complex. Under normal conditions, where mRNA translation is robust, mRNA decapping will occur on polyribosomes because it is kinetically faster than ribosomal run-off. We envision that decay in P-bodies is restricted to certain circumstances, such as under stress (when global translation is altered) or for mRNA populations in which ribosomal run-off is kinetically faster than mRNA decapping. Under conditions in which mRNA translational initiation or decapping are rate limiting (such as during stress), ribosomal run-off would predominate even in the absence of mRNA deadenylation and decay would be predicted to occur on ribosome-free mRNAs which may assemble into cytoplasmic P-bodies. Under normal conditions, however, we propose that remodeling of the 5′ UTR and deposition of the decapping complex is faster than the rate at which the mRNA can be cleared of ribosomes. In this regard, the mRNA is degraded co-translationally.
Perspectives Despite all that we have learned about mRNA decay in the past 20 years, there are still many mysteries. For instance, we still do not really understand how mRNA half-lives are determined for individual mRNAs. We are aware of elements that promote stability or instability, but by and large, is it unclear how these mRNA features really work. Perhaps central to this line of study is determining exactly how mRNA deadenylation rate is regulated. Deadenylation initiates mRNA decay and it clearly occurs at a message-specific rate, thereby setting the transcript’s
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overall half-life. Despite its importance, the control of mRNA deadenylation rate is still largely unexplored. We do know that mRNA deadenylation occurs on polyribosomes (Hilgers et al., 2006). As mentioned previously, altering the translation rate of a message greatly enhances deadenylation. It would be of great interest to explore how mRNA translation modulates mRNA deadenylation rate. Regarding decapping, this step of decay commits the message to be destroyed. Although a large collection of information on the decapping reaction has been gathered, such as characterization of the decapping enzyme, identification of global and message-specific decapping activators, and the potential interplay between the decapping enzyme and translation initiation factors, several outstanding and fundamental questions remain. For instance, the decapping complex is composed of more than 10 proteins in addition to the Dcp2/Dcp1 but we do not even have the simplest understanding of the temporal and special relationship of these factors during to each other and during the process of decay. Moreover, it has been documented that the same mRNA can have different stabilities under different environmental conditions (e.g., normal physiological conditions vs. stress conditions), thus determining how the decapping activity is regulated under different conditions will further provide insights into the regulation of mRNA decapping. Third, what is the role of P-bodies? P-bodies are evolutionarily conserved ribosome-free cellular foci with mRNA decay factors and certain mRNA decay intermediates. Interestingly, there is a reverse correlation between mRNA decay status and the formation and sizes of P-bodies under certain conditions (Franks and Lykke-Andersen, 2008; Parker and Sheth, 2007). It has been demonstrated by many different labs that P-bodies are not required for mRNA decay nor for translational repression (Chu and Rana, 2006; Decker et al., 2007; Eulalio et al., 2007; Sweet et al., 2007). The contribution, therefore, of P-bodies to mRNA metabolism is yet to be understood but of clear importance. Lastly, how is mRNA half-live determined in the context of on-going translation? Clearly subtle changes in messages translatability can lead to dramatic changes in the messages’ stability. For instance, dramatic disruption of translation events can elicit quality control pathways like nonsense-mediated mRNA decay (NMD), non-stop decay, or No-Go decay. It is important also to think about how subtile changes alter the way the normal pathway responds to the message. For instance, slowing initiation accelerates decay. Slowing elongation accelerates decay. Indeed, it appears that if translation is aberrant at any step, mRNA decay accelerates. Perhaps is it most appropriate to think of the normal degradation machinery as a monitor for the quality of protein synthesis events; its function is to respond when translation goes awry. mRNA are constantly associated with proteins, from its birth to death (reviewed in Moore, 2005). As we learn more, it seems that this mRNP has to be just right in order to facilitate mRNA metabolism events, like splicing, transport, and translation. It is tempting to speculate that changes to the mRNP occur during mRNA translation and this signals that the message is no longer translating efficiently and it is time for it to be cleared from the polyribosome pool. Perhaps the key event is deadenylation itself, which is a default reaction that
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dramatically impacts the mRNP structure and translatability of the mRNA. As the tail is shortened, translation rate changes and this is ‘sensed’ as a translational aberrancy. Once this occurs the messages is cleared from the cytoplasm by other decay factors. In this light, perhaps the normal degradation machinery becomes a critical aspect of monitoring the quality of gene expression as well as a regulator of its overall levels. Time will tell.
References Ashe, M.P., De Long, S.K., Sachs, A.B., 2000. Glucose depletion rapidly inhibits translation initiation in yeast. Molecular Biology of the Cell 11, 833–848. Bashkirov, V.I., Scherthan, H., Solinger, J.A., Buerstedde, J.M., Heyer, W.D., 1997. A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates. Journal of Cell Biology 136, 761–773. Beelman, C.A., Parker, R., 1994. Differential effects of translational inhibition in cis and in trans on the decay of the unstable yeast MFA2 mRNA. Journal of Biological Chemistry 269, 9687–9692. Beilharz, T.H., Humphreys, D.T., Clancy, J.L., et al., 2009. microRNA-mediated messenger RNA deadenylation contributes to translational repression in mammalian cells. PLoS One 4, e6783. Brengues, M., Teixeira, D., Parker, R., 2005. Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, 486–489. Caponigro, G., Muhlrad, D., Parker, R., 1993. A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: A stimulatory role for rare codons. Molecular and Cellular Biology 13, 5141–5148. Chu, C.Y., Rana, T.M., 2006. Translation repression in human cells by microRNAinduced gene silencing requires RCK/p54. PLoS Biology 4, e210. Coller, J., Parker, R., 2004. Eukaryotic mRNA decapping. Annual Review of Biochemistry 73, 861–890. Coller, J., Parker, R., 2005. General translational repression by activators of mRNA decapping. Cell 122, 875–886. Coller, J.M., Gray, N.K., Wickens, M.P., 1998. mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation. Genes & Development 12, 3226–3235. Coller, J.M., Tucker, M., Sheth, U., Valencia-Sanchez, M.A., Parker, R., 2001. The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes. RNA 7, 1717–1727. Cougot, N., Babajko, S., Seraphin, B., 2004. Cytoplasmic foci are sites of mRNA decay in human cells. Journal of Cell Biology 165, 31–40. Decker, C.J., Teixeira, D., Parker, R., 2007. Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae. Journal of Cell Biology 179, 437–449. Eulalio, A., Behm-Ansmant, I., Schweizer, D., Izaurralde, E., 2007. P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Molecular and Cellular Biology 27, 3970–3981. Eystathioy, T., Chan, E.K., Tenenbaum, S.A., et al., 2002. A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. Molecular Biology of the Cell 13, 1338–1351. Franks, T.M., Lykke-Andersen, J., 2008. The control of mRNA decapping and P-body formation. Molecular Cell 32, 605–615. Funakoshi, Y., Doi, Y., Hosoda, N., et al., 2007. Mechanism of mRNA deadenylation: Evidence for a molecular interplay between translation termination factor eRF3 and mRNA deadenylases. Genes & Development 21, 3135–3148. Giraldez, A.J., Mishima, Y., Rihel, J., et al., 2006. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–79. Goldstrohm, A.C., Wickens, M., 2008. Multifunctional deadenylase complexes diversify mRNA control. Nature Reviews 9, 337–344. Heikkinen, H.L., Llewellyn, S.A., Barnes, C.A., 2003. Initiation-mediated mRNA decay in yeast affects heat-shock mRNAs, and works through decapping and 5′-to-3′ hydrolysis. Nucleic Acids Research 31, 4006–4016. Hilgers, V., Teixeira, D., Parker, R., 2006. Translation-independent inhibition of mRNA deadenylation during stress in Saccharomyces cerevisiae. RNA 12, 1835–1845. Hosoda, N., Kobayashi, T., Uchida, N., et al., 2003. Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation. Journal of Biological Chemistry 278, 38287–38291.
Hu, W., Sweet, T.J., Chamnongpol, S., Baker, K.E., Coller, J., 2009. Co-translational mRNA decay in Saccharomyces cerevisiae. Nature 461, 225–229. Huarte, J., Stutz, A., O'Connell, M.L., et al., 1992. Transient translational silencing by reversible mRNA deadenylation. Cell 69, 1021–1030. Jacobson, A., Peltz, S.W., 1996. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annual Review of Biochemistry 65, 693–739. Kapp, L.D., Lorsch, J.R., 2004. The molecular mechanics of eukaryotic translation. Annual Review of Biochemistry 73, 657–704. Keeling, K.M., Salas-Marco, J., Osherovich, L.Z., Bedwell, D.M., 2006. Tpa1p is part of an mRNP complex that influences translation termination, mRNA deadenylation, and mRNA turnover in Saccharomyces cerevisiae. Molecular and Cellular Biology 26, 5237–5248. LaGrandeur, T., Parker, R., 1999. The cis acting sequences responsible for the differential decay of the unstable MFA2 and stable PGK1 transcripts in yeast include the context of the translational start codon. RNA 5, 420–433. Leung, A.K., Calabrese, J.M., Sharp, P.A., 2006. Quantitative analysis of Argonaute protein reveals microRNA-dependent localization to stress granules. Proceedings of the National Academy of Sciences of the United States of America 103, 18125–18130. Lykke-Andersen, J., 2002. Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay. Molecular and Cellular Biology 22, 8114–8121. Mangus, D.A., Jacobson, A., 1999. Linking mRNA turnover and translation: Assessing the polyribosomal association of mRNA decay factors and degradative intermediates. Methods 17, 28–37. Maroney, P.A., Yu, Y., Fisher, J., Nilsen, T.W., 2006. Evidence that microRNAs are associated with translating messenger RNAs in human cells. Nature Structural & Molecular Biology 13, 1102–1107. Moore, M.J., 2005. From birth to death: The complex lives of eukaryotic mRNAs. Science 309, 1514–1518. Muhlrad, D., Decker, C.J., Parker, R., 1995. Turnover mechanisms of the stable yeast PGK1 mRNA. Molecular and Cellular Biology 15, 2145–2156. Nottrott, S., Simard, M.J., Richter, J.D., 2006. Human let-7a miRNA blocks protein production on actively translating polyribosomes. Nature Structural & Molecular Biology 13, 1108–1114. Parker, R., Sheth, U., 2007. P bodies and the control of mRNA translation and degradation. Molecular Cell 25, 635–646. Pastori, R.L., Moskaitis, J.E., Buzek, S.W., Schoenberg, D.R., 1991. Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis. Molecular Endocrinology 5, 461–468. Peltz, S.W., Donahue, J.L., Jacobson, A., 1992. A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae. Molecular and Cellular Biology 12, 5778–5784. Rajyaguru, P., Parker, R., 2009. CGH-1 and the control of maternal mRNAs. Trends in Cell Biology 19, 24–28. Ross, J., 1995. mRNA stability in mammalian cells. Microbiological Reviews 59, 423–450. Scheuner, D., Song, B., McEwen, E., et al., 2001. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Molecular Cell 7, 1165–1176. Schoenberg, D.R., Maquat, L.E., 2009. Re-capping the message. Trends in Biochemical Sciences 34, 435–442. Schwartz, D.C., Parker, R., 1999. Mutations in translation initiation factors lead to increased rates of deadenylation and decapping of mRNAs in Saccharomyces cerevisiae. Molecular and Cellular Biology 19, 5247–5256. Schwartz, D.C., Parker, R., 2000. mRNA decapping in yeast requires dissociation of the cap binding protein, eukaryotic translation initiation factor 4E. Molecular and Cellular Biology 20, 7933–7942. Sheth, U., Parker, R., 2003. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808. Steiger, M., Carr-Schmid, A., Schwartz, D.C., Kiledjian, M., Parker, R., 2003. Analysis of recombinant yeast decapping enzyme. RNA 9, 231–238. Stevens, A., 2001. 5′-Exoribonuclease 1: Xrn1. Methods in Enzymology 342, 251–259. Sweet, T.J., Boyer, B., Hu, W., Baker, K.E., Coller, J., 2007. Microtubule disruption stimulates P-body formation. RNA 13, 493–502. Theodorakis, N.G., Cleveland, D.W., 1992. Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation. Molecular and Cellular Biology 12, 791–799. Thompson, S.R., Goodwin, E.B., Wickens, M., 2000. Rapid deadenylation and Poly (A)-dependent translational repression mediated by the Caenorhabditis elegans tra-2 3′ untranslated region in Xenopus embryos. Molecular and Cellular Biology 20, 2129–2137.
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Tritschler, F., Huntzinger, E., Izaurralde, E., 2010. Role of GW182 proteins and PABPC1 in the miRNA pathway: A sense of deja vu. Nature Reviews 11, 379–384. Wang, Y., Liu, C.L., Storey, J.D., et al., 2002a. Precision and functional specificity in mRNA decay. Proceedings of the National Academy of Sciences of the United States of America 99, 5860–5865.
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Wang, Z., Jiao, X., Carr-Schmid, A., Kiledjian, M., 2002b. The hDcp2 protein is a mammalian mRNA decapping enzyme. Proceedings of the National Academy of Sciences of the United States of America 99, 12663–12668. Yamashita, A., Chang, T.C., Yamashita, Y., et al., 2005. Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover. Nature Structural & Molecular Biology 12, 1054–1063.
The destruction of mRNA is essential for proper regulation of gene expression. All mRNA must be destroyed to ensure that nuclear regulatory decisions are manifested as cytoplasmic events. In other words, ceasing mRNA transcription in response to environmental or physiological cues is irrelevant as long as cytoplasmic mRNA exists and is translated. From this, it can be said that mRNA decay contributes significantly to the proteomes’ overall architecture by allowing cells to quickly adapt to environmental and physiological changes. Although much is known about mRNA decay in eukaryotes, understanding how the process is modulated is far from complete. In this section, we first discuss our current understanding of eukaryotic mRNA decay. Then we put mRNA degradation in the context of mRNA translation, which is another important cyctoplasmic event of gene expression, as these two events are known to be tightly integrated with each other for decades. In the last few years, many studies have concentrated on this relationship as a means to elucidate regulation of mRNA degradation. From this, two views have arisen. The first suggests that mRNA must be removed from ribosomes to be destroyed in ribosome-free areas, such as P-bodies. The other view proposes mRNA decay occurs in concert with protein synthesis. Here we summarize our current understanding of how eukaryotic mRNA decay interconnects with mRNA translation, and then analyze the data that have lead to these two diametrically opposed models of where mRNA decay occurs within the cell. Finally, we attempt to reconcile these two views and suggest important areas for future investigation.
Overview of Eukaryotic mRNA turnover Cytoplasmic mRNAs are destroyed by two major pathways. Both are initiated by the exonucleolytic digestion of the 3′ poly (A) tail (a process termed deadenylation). Deadenylation is a key regulatory step in mRNA half-life determination because its rate is highly variable among transcripts. In some cases, deadenylation leads to mRNA destruction 3′–5′ by the cytoplasmic exosome. In other cases, deadenylation causes the rapid removal of the 5′ 7mGpppN cap and digestion of the transcript body 5′–3′ (reviewed in Coller and Parker, 2004). Which pathway predominates is not clearly understood. Most likely transcript, tissue, and species specific differences dictate the predominate pathway employed. In this article, we focus exclusively on the deadenylation-dependent decapping pathway since it has been extensively characterized and its relationship to translation is more established than that of the cytoplasmic exosome-dependent pathway. As mentioned, deadenylation is the first step of mRNA decay and is often thought to be rate limiting. This reaction is carried out by several enzymes (deadenylases) that digest the poly(A) tail in a 3′–5′ direction. Generally, mRNA deadenylation is biphasic, the first step is initiated by the deadenylase dependent
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poly(A) nuclease 2 (PAN2). Following this initial trimming event, the bulk of the tail is digested by the enzyme complex CCR4/POP2/NOT (Yamashita et al., 2005). Other deadenylases, however, exist including poly(A)-specific ribonuclease (PARN), Nocturin, and Angel proteins (reviewed in Goldstrohm and Wickens, 2008). It would appear, then, that deadenylation is facilitated by a seemingly redundant class of enzymes. Diversity in transcript stability might occur in part by the manner in which these enzymes achieve transcript specificity. For example, 3′UTR-binding proteins and miRNAs can enhance deadenylation by tethering a specific enzyme (Giraldez et al., 2006). In yeast as well as in mammalian cells, deadenylation usually leads to destruction of the mRNA. In some systems, deadenylation can lead to translational storage of the mRNA. Although still unclear, it is thought that the poly(A) tail mediates its effect on mRNA translation and decay by way of an interaction between the poly(A)-binding protein (PAB) and the translation initiation factor eIF4G (Coller et al., 1998; Jacobson and Peltz, 1996). Deadenylation serves to displace PAB from the mRNA thereby liberating its protective and/or translational stimulatory function. In the cases where transcript destruction follows deadenylation, once the mRNA tail has been trimmed to a length no longer supporting PAB binding, the 7-methyl-guanosine cap structure is rapidly cleaved from the mRNA’s 5′ end. The decapping reaction requires the enzymatic activity of the DCP2 protein. Enzymatically, the pyrophosphatase activity of DCP2 cleaves the alpha-beta bond of the 5′ cap liberating m7′GDP and leaving a 5′ monophosphate on the transcript. In vitro, DCP2 is sufficient for this activity (Lykke-Andersen, 2002). Although simple in chemistry in vitro, the in vivo orchestration of mRNA decapping requires a suite of proteins including DCP1, DHH1(RCK/p54), Hedls (only in metazoans), Pat1p (P100), Lsm1–7, Lsm12, Scd6 (Trailerhitch...), and Edc1–3 (reviewed in Franks and Lykke-Andersen, 2008). The precise function(s) of these factors is murky. Some decapping activators like LSM1–7 and Hedls may serve structural roles in promoting binding of DCP2 to the mRNA. Others like DHH1 and PAT1 are hypothesized to promote DCP2’s accessibility to the cap by dissociating factors bound at the mRNA 5′ end, such as eIF4F (Coller and Parker, 2005; Chu and Rana, 2006). Irrespective of their true function, the complexity of this process indicates that mRNA decapping is tightly regulated. Control of decapping makes sense since this step of decay commits the mRNA to destruction and unlike deadenylation is largely irreversible although exceptions have been documented (reviewed in Schoenberg and Maquat, 2009). Subsequent to decapping, the transcript is quickly and efficiently destroyed by the 5′–3′ exonuclease, XRN1. This enzyme is highly processive and decay intermediates are hard to detect (Stevens, 2001). To date, no regulation of XRN1 activity has been detected, thus, it is generally thought that the 5′–3′ exonucleolytic decay is not a rate-limiting step in turnover.
Encyclopedia of Cell Biology, Volume 1
doi:10.1016/B978-0-12-394447-4.10057-4
The Interplay between Eukaryotic mRNA Degradation and Translation
mRNA Degradation is Intimately Linked to Translation All mRNA must and will succumb to decay. Transcript decay is, therefore, a default state. The spectrum of observed half-lives most likely represents acceleration or deceleration of this default rate. The translatability of an mRNA is a critical contributor to its overall stability (reviewed in Franks and Lykke-Andersen, 2008; Jacobson and Peltz, 1996). Specifically, an inverse correlation has been established showing efficiently translated mRNAs have longer half-lives; while poorly translated mRNAs are unstable. These findings suggest that a major determinant of mRNA stability is the translation machinery. Curiously, however, modulation of each specific sub-step of translation, i.e., initiation, elongation, or termination, is perceived to have disparate effects on mRNA’s half-life. It is important to note that this perception is based largely on the manner of analysis (see below). The when and where of mRNA decapping and exonucleolytic decay has been the subject of some debate. Models for where mRNA decay takes place are in part based on the distinct manner in which translational inhibition affects mRNA decay. Experimental evidence has suggested two possibilities. On one side data seems to indicate that before decapping can occur, the translational machinery must be liberated and then the message is destroyed in specialized places within the cell. On the other side, there are data to suggest that mRNA never leave polysomes and are degraded co-translationally. In the following sections we highlight the work of many individuals whose observations have generated these two distinct views.
The Ying-Yang of Translational Initiation and Transcript Stability Translational initiation is posited to be in direct competition with mRNA decapping (reviewed in Franks and Lykke-Andersen, 2008). From a priori view, this hypothesis makes sense because both translational initiation and mRNA decapping are dependent on factors binding on or near the 5′ cap (Cougot et al., 2004). This theory is also supported by experimental data. In a simplified view, translation initiation consists of three major events: first recognition of the cap by eIF4F, which is composed of eIF4E, eIF4G, and eIF4A; second, recruitment of the 40 S ribosomal subunit; third, scanning of the 40 S ribosomal subunit to the first AUG (reviewed in Kapp and Lorsch, 2004). Altering the rate of any of these three events has a profound effect on mRNA half-live. For instance, mutations in eIF4F reducing its affinity for the cap result in rapid and efficient deadenylation and decapping (Schwartz and Parker, 1999). Similarly, inhibiting 40 S ribosomal subunit recruitment and scanning accelerates mRNA deadenylation and decapping. For example, inactivation of eIF3, thereby blocking 40 S recruitment (Kapp and Lorsch, 2004), results in rapid mRNA decay (Heikkinen et al., 2003). Cis-acting RNA structures that inhibit 40 S ribosomal subunit scanning also accelerate mRNA turnover (Muhlrad et al., 1995; Coller and Parker, 2005). Lastly, the context of the AUG start codon influences mRNA stability. Specifically, nucleotide changes around the translation start codon predicted to decrease translational efficiency also dramatically destabilize reporter transcripts (LaGrandeur and
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Parker, 1999). Taken together these correlations between translational initiation and mRNA decapping predict that decreasing ribosome occupancy on the 5′UTR allows the decay machinery to associate more efficiently with the transcript. Moreover, these data suggest that the translation initiation factors and decapping factors vie for the mRNA in vivo. A direct competition between decapping and eIF4E has been documented in vitro. Purification of DCP1 from yeast extracts allows decapping to be monitored in vitro (presumable because purification of DCP1 brings along the decapping enzyme DCP2). Addition of recombinant eIF4E protein efficiently inhibits in vitro decapping mediated by the purified decapping enzyme (Schwartz and Parker, 2000). In conjunction with the aforementioned in vivo work, these data have led to the model that dissociation of eIF4E is required before mRNA decapping can occur. Importantly, this notion is supported by genetic analysis. Specifically, loss of eIF4E will restore the decapping activity when it is partially impaired genetically (i.e., DCP1Ts; Schwartz and Parker, 2000). Collectively, these results indicate that the mRNA decapping enzyme (Dcp2/Dcp1) and eIF4E are probably in competition for the 5′-end cap structure on an mRNA. Clearly, mRNA decapping is stimulated by loss of eIF4E function, but it is also important to note that in vivo inhibition of translational initiation in any manner also greatly enhances deadenylation rate. The effects of translation rate on deadenylation have remained unexplored. Undoubtedly there is a positive correlation between the efficiency of translational initiation and mRNA stability. The displacement of eIF4E from the cap, therefore, is proposed to be a necessary first step in mRNA decapping (reviewed in Coller and Parker, 2004). At this point, it is important to note that the inverse correlation between translation rate and mRNA decapping does not hold true under every physiological condition. For example, cellular stress can have profound effects on translational initiation and mRNA decay. Under these extreme conditions the predictable correlation between translation and decay does not occur. Specifically, translational initiation is inhibited when cells are deprived of glucose (Ashe et al., 2000). Despite this, mRNAs are dramatically stabilized rather than destabilized (Hilgers et al., 2006). On the other hand, amino acid deprivation also powerfully inhibits translation initiation (ref) but mRNAs are greatly destabilized under this condition. Lastly, the unfolded protein response also triggers repression of translation (Scheuner et al., 2001), but this stress does not affect mRNA stability at all (Hilgers et al., 2006). One possible explanation for these discrepancies is that each distinct stress alters one or more of the mRNA decay factors. Alternatively, each stress might change mRNP dynamics and therefore the manner by which it is perceived by the decay machinery. It seems likely, therefore, that a more detailed understanding of how cellular stress impacts mRNA stability will shed more light on the complex interplay between decay and translation.
Translation Elongation and mRNA Turnover It is postulated that once an mRNA is engaged in elongation, ribosomes provide a protective quality that insulates the
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transcript from decay (Jacobson and Peltz, 1996). This theory, however, is controversial. Unlike translational initiation where slowing the process enhances decay, the manner in which elongation is perturbed dramatically influences the result obtained. For example, treating cells with the translation elongation inhibitor, cycloheximide (a drug that freezes ribosomes on mRNA), results in a significant inhibition of decay in budding yeast and high eukaryotes (Beelman and Parker, 1994; Ross, 1995). Similar results are obtained when elongation is stopped using genetic mutation. Specifically, depleting charged tRNAs by inhibiting maturation of their 3′ end will dramatically stabilize mRNA (Peltz et al., 1992). In both cases, blocking elongation is suggested to inhibit decapping (Beelman and Parker, 1994), deadenylation still occurs, albeit at a slightly reduced rate (Hilgers et al., 2006). It is important to point out, that both of these procedures do not kinetically slow elongation, rather, they freeze ribosomes on mRNA and block elongation altogether. Paradoxically, slowing the rate of translation elongation is similar to slowing translational initiation in that it destabilizes mRNAs. For example, rare codons are recognized by tRNAs with low concentrations within the cell, thus, mRNAs with rare codons have a relatively slow translation rate compared to the mRNAs with cognate normal codons. The presence of rare codons within an mRNA saturates the transcript with ribosomes and dramatically shortens halflife (Caponigro et al., 1993; Hu et al., 2009). Slowing ribosome translocation in this manner, therefore, destabilizes mRNAs. One possible explanation for these differences is that freezing mRNA on ribosomes may ‘lock’ the transcript in an RNP conformation that is resistant to mRNA decay factors. Conversely slowing elongation enhances decay by a yet unknown mechanism.
Translation Termination and mRNA Stability Eukaryotic mRNA translation termination requires two release factors, eRF1 and eRF3. Translation termination process can influence mRNA half-life. Specifically, it was observed that the N-terminal domain of eRF3, which is not required for translation termination, can interact with Pab1, and this interaction is involved in modulating mRNA stability (Hosoda et al., 2003). Disruption of this interaction results in translationdependent stabilization of mRNA caused by decreased deadenylation rate (Hosoda et al., 2003). Interestingly, it was further found that certain deadenylase complexes can also bind to the same site on Pab1 that is involved in the interaction with eRF3 (Funakoshi et al., 2007). Thus, it has been postulated that eRF3 can regulate mRNA deadenylation by competitively binding to the Pab1, which then modulates the recruitment and activation of deadenylase complexes (Funakoshi et al., 2007). In addition to the release factors, other proteins that can modulate translation termination can also influence mRNA stability. For example, a recent characterized protein named Tpa1 can interact with the two release factors and regulate the readthrough of stop codons (Keeling et al., 2006). Interestingly, although the detailed mechanisms still remain elusive, knocking out this protein can have decreased deadenylation rate and increased mRNA stability (Keeling et al., 2006). Collectively, these results suggest that
mRNA translation termination can results in mRNP conformational changes that can influence mRNA stability, likely via modulating mRNA deadenylation.
Where Does Eukaryotic mRNA Degradation Occur Within the Cell? In recent years, understanding where mRNA degradation occurs within the cell has been given a great deal of scientific investigation. The thought is that insight into how the large repertoire of observed mRNA half-lives is achieved may come from knowing when and where degradation occurs within the cytoplasm. At present, there are two different views on where mRNA degradation takes place. The first comes from the discovery that mRNA decapping and decay factors aggregate into punctuate, microscopically visible structures. These structures have been given the epitaph of processing bodies or P-bodies for short. Importantly, P-bodies do not contain ribosomes nor do they contain most translational initiation factors. What they do contain is the full complement of decapping proteins, certain mRNA decay intermediates, and in higher eukaryotes, the miRNA machinery (reviewed in Franks and Lykke-Andersen, 2008; Parker and Sheth, 2007). In conjunction with the aforementioned findings that mRNA translation is generally positively correlated with half-life, these data have led to a popular ‘two-step’ model for regulating transcript stability in which ribosome dissociation first occurs, then the mRNA is trafficked to P-bodies where mRNA decapping ensues (reviewed in Franks and Lykke-Andersen, 2008; Parker and Sheth, 2007). We refer to this model as the ‘mRNA cycle hypothesis,’ because it is postulated that mRNA can cycle in and out of polyribosomes and into a quiescent state that can be either stored/reutilized or degraded (Brengues et al., 2005). A second model supported in the literature, is that mRNA decay does not involve a fundamental transition between a polyribosomebound state and a translationally repressed state, but rather occurs co-translationally (Hu et al., 2009). This hypothesis dates back almost 20 years (Beelman and Parker, 1994; Mangus and Jacobson, 1999), but has lacked strong experimental evidence until recently. Nonetheless, both models are supported but have their limitations. Here, we will examine these two models in detail and at the end attempt to provide a unifying theory.
The mRNA Cycle Hypothesis – From Polysome to p-Bodies (and Perhaps Back) The theory that translation and decay are partitioned events is based in part on the aforementioned distinct effects translation initiation versus elongation have on mRNA half-life. The mRNA cycle hypothesis suggests that mRNA is translated and then at some point becomes translationally silenced and moved into a state of quiescence. Once this inactive state is achieved, the mRNA can either be destroyed or perhaps reutilized in response to certain cues. In theory, translational repression of mRNA makes sense from a logistical stand point. The 5′-cap structure is occupied by a suite of proteins that promote 40 S ribosome joining. At some
The Interplay between Eukaryotic mRNA Degradation and Translation
point, DCP2 must have access to the cap and 5′UTR, and thus it seems logical that the 5′ end is remodeled and initiation factors displaced (Steiger et al., 2003). It is predicted that the downstream consequence of this remodeling must be translational repression, as manifest by ribosome dissociation. The RNA cycle hypothesis is rooted in studies of translational initiation and its effect on decay. As discussed in the previous section, Schwartz and Parker (1999) found that mutations in eIF4F result in mRNA destabilization. Cap binding by eIF4F, therefore was proposed to be in competition with mRNA decapping. Similar results were seen when other aspects of initiation were compromised, including 40 S joining and AUG recognition. Moreover, in vitro experiments demonstrated that addition of purified eIF-4E could reduce decapping in cell extracts (Schwartz and Parker, 2000). The combined findings were interpreted as a direct competition between eIF4E and DCP2 exists. A corollary of this hypothesis is that translational repression, defined as ribosome dissociation, is required before mRNA decapping. The mRNA cycle model is also supported by the discovery that several decapping regulators influence mRNA translation. Specifically, DHH1 and PAT1 were shown to be bona fide translational repressors in yeast and humans (Coller and Parker, 2005; Chu and Rana, 2006). Consistent with the notion, DHH1 is homologous to factors implicated in maternal mRNA storage (reviewed in Rajyaguru and Parker, 2009) thus decapping was hypothesized to be similar to posttranscriptional events that occur during early development (Coller et al., 2001). The nature of how mRNA decapping regulators influence mRNA translation is still unknown, but has been proposed to inhibit translation initiation (Coller and Parker, 2005). Other aspects of mRNA decay are also thought to result in translational silencing, and possible ribosome dissociation. Specifically, mRNA deadenylation can lead to translational repression (Huarte et al., 1992). This is especially well documented during development. The poly(A) tail and its binding protein, PAB, are hypothesized to interact with the 5′ cap via a protein interaction with eIF4G. Poly(A) is popularly believed to stimulate translation by way of this ‘close-loop’ (Jacobson and Peltz, 1996). Collectively, the first step of mRNA decay, deadenylation, would be predicted to result in a loss of translational efficiency. It is important to note, however, that controversy exists whether deadenylation is a cause or effect of translational repression in some context. P-bodies, above all things, bolstered the notion that ribosome dissociation is required for mRNA decapping and that these events are similar to storage events that occur in other contexts (reviewed in Parker and Sheth, 2007; Rajyaguru and Parker, 2009). The discovery of P-bodies dates back to 1997 when Bashkirov et al. (1997) cloned the mouse homolog of the known 5′–3′ exonuclease Xrn1. Using mouse E10 fibroblast cells, it was observed that mXrn1 localized to punctuate cytoplasmic structures. At this time, the authors proposed that these structures may represent either sites for RNA turnover or sites in which the enzyme is stored until used. P-bodies were rediscovered in 2002 when Eystathioy et al. (2002) found that the human auto-antigen protein, GW182, was also found in cytoplasmic granules. Although it was not clear at this time,
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GW182 is a critical factor in mediating miRNA translational control (Tritschler et al., 2010). One year later, a flurry of studies simultaneously demonstrated that, in the addition to XRN1, the major decapping factors also are found in cytoplasmic foci (many refs). Sheth and Parker (2003) extended this analysis by showing that mRNA decay intermediates co-localized with these granules in a manner that is dependent on the enzymatic activity of yeast XRN1. Similar results were seen in humans (Cougot et al., 2004). Thus the aggregation of decay factors in granules was suggested to be of functional significance and that these foci represented the place where mRNAs were degraded. P-bodies are devoid of ribosomes and other vital translational initiation factors (Sheth and Parker, 2003; Cougot et al., 2004). Coupled with the aforementioned results indicating decapping is in competition with translational initiation, it was further proposed that mRNA must be removed from ribosomes and trafficked to a P-body before decay ensues (reviewed in Franks and Lykke-Andersen, 2008). Indeed the very term P-bodies, or processing bodies was coined, in part, to draw similarity to P-granules or polar bodies, known sites of mRNA storage in germ-lines, oocytes, and embryos (reviewed in Parker and Sheth, 2007; Rajyaguru and Parker, 2009). Consistent with this, P-bodies have also been suggested to contain translational quiescent mRNA (Brengues et al., 2005; Parker and Sheth, 2007; Franks and LykkeAndersen, 2008). In mammalian systems, the miRNA machinery co-localizes to P-bodies further supporting a model in which P-bodies are sites of mRNA storage. In yeast, stress conditions like glucose deprivation result in global shut-down of mRNA translation and mRNA decay. This correlates with an increase in P-body size and abundance. An increase in P-body size has been interpreted as an influx of mRNA into these structures (Brengues et al., 2005; Sheth and Parker, 2003). Taken together, mRNA decapping has hypothesized to require removal of mRNA from the translational apparatus followed by packaging into P-bodies where either decay or storage ensues. The RNA cycle model is very provocative, but there are still many questions left to be answered. For instance, one important feature of this hypothesis is that DCP2 and eIF-4E are in direct competition with each other for cap access. While this competition is clear in vitro, in vivo the relationship between DCP2 and eIF4E is more complex. Specifically, if a simple competitive relationship existed, then loss of eIF4E function would result in promiscuous mRNA decapping independent of deadenylation. Although perturbations in initiation result in accelerated decapping, in all cases when initiation is impaired (i.e., initiation factor mutants, AUG context changes, and cis-acting initiation blocks) decapping is not uncoupled from deadenylation. Indeed, loss of deadenylation by ccr4 mutation stabilizes non-translating mRNAs. Rather, loss of eIF-4F function results in rapid deadenylation followed by rapid decapping (Schwartz and Parker, 1999). These data indicate that loss of eIF-4E is not sufficient to elicit decapping directly, rather dissolution of the initiation complex sensitizes the mRNA toward decay in a fashion that is still unknown. In this light, the notion that eIF-4E displacement is a critical event in decapping stimulated by deadenylation requires reinvestigation.
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The RNA cycle hypothesis is also based on the notion that removing mRNAs from ribosomes accelerates their decay. Based on this, polysomes would provide a protective quality to the transcript, for example, sequestering the message from P-bodies. While generally, impeding translational initiation promotes rapid mRNA decay, important caveats exist. For instance, there is no correlation between changes of translation status and mRNA stability under conditions of stress known to impede protein synthesis (see above). Moreover, little evidence exists that ribosomes protect the mRNA from destruction. In fact, slowing translation elongation by use of rare codons saturates the message with ribosomes, but, paradoxically, greatly enhances its decay by the normal deadenylation-dependent decapping machinery. It appears then that an mRNA that never has ribosomes is degraded quickly, and an mRNA that is saturated with slowly moving ribosomes is also degraded quickly. Thus, there is no obvious need for the mRNA to dissociate from the translational machinery in order to be degraded efficiently (see below). Lastly, an important aspect of the RNA cycle hypothesis is that once mRNA leaves ribosomes they enter P-bodies. From a quantitative point of view, it is still unclear to what extend mRNA decapping and 5′–3′ exonucleolytic decay occur in ribosome-free areas in the cell. Moreover, it is unclear how much of the decapping complex assembles into P-bodies versus the soluble cytosol. In some cases, it has been suggested to be only a minor fraction. For instance quantification of the P-body protein Ago2 in mammalian cells revealed that less than 5% of total Ago2 is localized in P-bodies (Leung et al., 2006). Second, P-bodies, whose abundance correlates well with events that enhance translation repression and mRNA decay, are not required for either of these events. Specifically, in both yeast and higher eukaryotes, several groups have shown that P-body formation can be uncoupled from mRNA turnover and translation repression (Sweet et al., 2007; Decker et al., 2007; Eulalio et al., 2007). Together, these data demonstrate that the function of P-bodies remains enigmatic. Clearly, however, the aggregation of decapping factors into P-bodies is not required for normal rates of mRNA turnover. In summary, the RNA cycle hypothesis is provocative. It provides a simple explanation for how mRNA turnover and translation are interconnected. Importantly, however, significant caveats exist suggesting that mRNA turnover is not as simplistic as the model suggest.
Parting on Polysomes A second model that has emerged in the literature is that message decay takes place co-translationally. In other words, mRNA decapping and 5′–3′ exonucleolytic digestion occur primarily on polyribosomes and not in a ribosome-free state (e.g., a P-body). The hypothesis that mRNAs are destroyed on polysomes is not new. In most cases, however, this scenario has only been observed in special cases; specifically albumin and β-tubulin mRNA (Pastori et al., 1991; Theodorakis and Cleveland, 1992). Nonetheless, small clues are seen throughout the literature. Early evidence was first seen yeast. As mentioned in previous sections, treating yeast cells with the translational elongation inhibitor cycloheximide leads to the
dramatic stabilization of mRNAs. Recent interpretations of this effect are that the mRNA is sequestered in polyribosomes under these conditions and away from P-bodies (reviewed in Parker and Sheth, 2007; Franks and Lykke-Andersen, 2008). Interestingly, however, Beelman and Parker (1994) investigated mRNA decay in cells treated with cycloheximide and found the mRNA accumulated as a slightly shorter species over time. Although this finding wasn’t further characterized, they proposed the truncated mRNA was the result of decapping and digestion up to a ribosome stalled at the AUG. Consistent with the hypothesis of co-translational decapping/decay, it was also found that in humans decapping activity co-sediments with polysomes (Wang et al., 2002a,b). Mangus and Jacobson (1999) observed that decay intermediates similar to those observed in P-bodies were also associated with polyribosomes. Lastly, we provided additional supportive by showing that the majority of decapping is observed while the transcript is saturated with ribosomes (Hu et al., 2009). Collectively, these data argue that under normal physiological conditions, mRNA decapping and 5′–3′ exonucleolytic digestion occur on polyribosomes. A corollary of this hypothesis is that a transition into a translational repressed state, a predicted by the RNA cycle hypothesis, is not required for efficient mRNA decay. Like the RNA cycle hypothesis, the model of co-translational decay also has important caveats to mention. First, numerous reports have shown that events like deadenylation inhibit protein synthesis (Huarte et al., 1992; Thompson et al., 2000; Beilharz et al., 2009), yet no apparent difference in ribosome association can be seen for mRNA before and after deadenylation in our analysis. It is important to note here that polysome analysis provides direct physical evidence for the binding of ribosomes to an mRNA, it does not provide any information about translation rate. For example, it has been documented that miRNA targets are saturated with ribosomes, yet no protein output is detectable (Maroney et al., 2006; Nottrott et al., 2006). It is unclear how this is possible, but most likely indicates that there are enigmatic mechanisms of translational control that have yet to be revealed. Indeed in our own analysis we cannot conclude if nonadenylated mRNAs are translating at the same rate as fully adenylated mRNA. A clear and important area of future investigation is to correlate ribosome occupancy with adenylation status and protein output. Perhaps this analysis will shed light on this unexpected finding. Second, it is clear that the decapping regulators DHH1 and PAT1 inhibit protein synthesis most likely by altering translational initiation (Coller and Parker, 2005) and that mutations in initiation factors can accelerate decapping. In this regard, it is important to realize that polysome analysis is a gross measurement of ribosome-associated mRNPs. Subtle changes such as rearrangement of the mRNA's 5′ UTR are beyond the detection limit of this assay, unless they result in dramatic changes in polysome association. Lastly, the contribution of initiation factors to polyribosome association/maintenance is still unclear. In recent years it has been observed that loss of initiation factor function in vivo has only a mild effect on polyribosome formation. Thus, it is formally possible that 5′UTR remodeling can occur with little or no appreciable change in ribosome occupancy. The explanation for this paradox remains unclear, but might indicate that initiation factors are only rate limiting for
The Interplay between Eukaryotic mRNA Degradation and Translation
translation under certain circumstances or during early events in protein synthesis before the establishment of polyribosomes.
What Does It All Mean? The interplay between translation and message decay is of great importance for understanding the overall regulation of gene expression. As discussed in the previous two sections, there are two points of view. One is that most mRNA decay occurs after ribosome dissociation and perhaps in P-bodies. The other suggests that mRNA never leave ribosomes and decay is co-translational. Both hypothesizes are based on clear experimental observations. Nether model is perfect and both have important caveats for consideration. In the end, the truth most likely lies somewhere in between. Biochemical data indicate that polyribosomes represent a major site of mRNA decapping within the cell under normal growth conditions. Importantly, however, evidence also exists to suggest that decay occurs, at some level, in P-bodies. We propose that all mRNA degradation is initiated on polyribosomes at the step of deadenylation. Where subsequent transcript degradation occurs is a function of the messages’ translation rate versus relative to its decapping rate. We hypothesize that deadenylation does indeed reduces translational efficiency (Huarte et al., 1992) perhaps through loss of the PAB, Pab1p (Jacobson and Peltz, 1996), or association of decapping regulators such as DHH1 and PAT1 (Coller and Parker, 2005). These events allow for the remodeling of the 5′UTR and deposition of a decapping complex. Under normal conditions, where mRNA translation is robust, mRNA decapping will occur on polyribosomes because it is kinetically faster than ribosomal run-off. We envision that decay in P-bodies is restricted to certain circumstances, such as under stress (when global translation is altered) or for mRNA populations in which ribosomal run-off is kinetically faster than mRNA decapping. Under conditions in which mRNA translational initiation or decapping are rate limiting (such as during stress), ribosomal run-off would predominate even in the absence of mRNA deadenylation and decay would be predicted to occur on ribosome-free mRNAs which may assemble into cytoplasmic P-bodies. Under normal conditions, however, we propose that remodeling of the 5′ UTR and deposition of the decapping complex is faster than the rate at which the mRNA can be cleared of ribosomes. In this regard, the mRNA is degraded co-translationally.
Perspectives Despite all that we have learned about mRNA decay in the past 20 years, there are still many mysteries. For instance, we still do not really understand how mRNA half-lives are determined for individual mRNAs. We are aware of elements that promote stability or instability, but by and large, is it unclear how these mRNA features really work. Perhaps central to this line of study is determining exactly how mRNA deadenylation rate is regulated. Deadenylation initiates mRNA decay and it clearly occurs at a message-specific rate, thereby setting the transcript’s
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overall half-life. Despite its importance, the control of mRNA deadenylation rate is still largely unexplored. We do know that mRNA deadenylation occurs on polyribosomes (Hilgers et al., 2006). As mentioned previously, altering the translation rate of a message greatly enhances deadenylation. It would be of great interest to explore how mRNA translation modulates mRNA deadenylation rate. Regarding decapping, this step of decay commits the message to be destroyed. Although a large collection of information on the decapping reaction has been gathered, such as characterization of the decapping enzyme, identification of global and message-specific decapping activators, and the potential interplay between the decapping enzyme and translation initiation factors, several outstanding and fundamental questions remain. For instance, the decapping complex is composed of more than 10 proteins in addition to the Dcp2/Dcp1 but we do not even have the simplest understanding of the temporal and special relationship of these factors during to each other and during the process of decay. Moreover, it has been documented that the same mRNA can have different stabilities under different environmental conditions (e.g., normal physiological conditions vs. stress conditions), thus determining how the decapping activity is regulated under different conditions will further provide insights into the regulation of mRNA decapping. Third, what is the role of P-bodies? P-bodies are evolutionarily conserved ribosome-free cellular foci with mRNA decay factors and certain mRNA decay intermediates. Interestingly, there is a reverse correlation between mRNA decay status and the formation and sizes of P-bodies under certain conditions (Franks and Lykke-Andersen, 2008; Parker and Sheth, 2007). It has been demonstrated by many different labs that P-bodies are not required for mRNA decay nor for translational repression (Chu and Rana, 2006; Decker et al., 2007; Eulalio et al., 2007; Sweet et al., 2007). The contribution, therefore, of P-bodies to mRNA metabolism is yet to be understood but of clear importance. Lastly, how is mRNA half-live determined in the context of on-going translation? Clearly subtle changes in messages translatability can lead to dramatic changes in the messages’ stability. For instance, dramatic disruption of translation events can elicit quality control pathways like nonsense-mediated mRNA decay (NMD), non-stop decay, or No-Go decay. It is important also to think about how subtile changes alter the way the normal pathway responds to the message. For instance, slowing initiation accelerates decay. Slowing elongation accelerates decay. Indeed, it appears that if translation is aberrant at any step, mRNA decay accelerates. Perhaps is it most appropriate to think of the normal degradation machinery as a monitor for the quality of protein synthesis events; its function is to respond when translation goes awry. mRNA are constantly associated with proteins, from its birth to death (reviewed in Moore, 2005). As we learn more, it seems that this mRNP has to be just right in order to facilitate mRNA metabolism events, like splicing, transport, and translation. It is tempting to speculate that changes to the mRNP occur during mRNA translation and this signals that the message is no longer translating efficiently and it is time for it to be cleared from the polyribosome pool. Perhaps the key event is deadenylation itself, which is a default reaction that
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dramatically impacts the mRNP structure and translatability of the mRNA. As the tail is shortened, translation rate changes and this is ‘sensed’ as a translational aberrancy. Once this occurs the messages is cleared from the cytoplasm by other decay factors. In this light, perhaps the normal degradation machinery becomes a critical aspect of monitoring the quality of gene expression as well as a regulator of its overall levels. Time will tell.
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