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Messenger RNA degradation in eukaryotes
Cell, Vol. 74, 413-421, August 13, 1993, Copyright © 1993 by Cell Press
Review
Messenger RNA Degradation in Eukaryotes Alan B, Sachs Division of Biochemistry and Molecular Biology University of California, Berkeley Berkeley, California 94720
Introduction Messenger RNA (mRNA) degradation in eukaryotic cells is a regulated process that can determine the level of expression of a gene. In particular, the existence of highly unstable RNAs allows for rapid and precise reductions or elevations in transcript levels following changes in transcript production rates or alterations in the machinery dedicated to transcript degradation. The mechanisms by which mRNAs are degraded and the sequence elements within the mRNAs that determine their stability are the subject of this review. A more detailed treatment of this topic and of prokaryotic mRNA degradation can be found in a recent collection of reviews (Belasco and Brawerman, 1993). The range of mRNA stability in eukaryotic cells can vary over several orders of magnitude (reviewed in Peltz et al., 1991). For instance, in higher cells some mRNAs are degraded with a half-life of about 20 min, while in the same cell, other mRNAs are degraded with half-lives over 24 hr. Similar differences in mRNA half-lives have been found in yeast, with the most unstable messages decaying in less than 5 min and the most stable decaying at rates slower than 60 min (Herrick et al., 1990). Searches for sequence elements on mRNA that modulate decay rates have revealed several types of destabilizing elements (see below). These elements are identified by their ability to confer destabilization to a normally stable message. Interestingly, removal of these sequences from their native context often does not lead to stabilization of that message. This has been interpreted to suggest that mRNAs have multiple destabilizing sequences and that the process by which mRNA is targeted to decay may be redundant within the message. The search for mRNA stabilizing sequences has been much less successful. In some cases, sequence elements that silence destabilizing sequences have been found (see below), but in no case has a sequence by itself been found to confer stability to another mRNA. The exceptions to this are regions of RNA secondary structure that can stabilize mRNA fragments produced during the decay reaction (Decker and Parker, 1993; Vreken and Rau6, 1992) and the cap and poly(A) tail of the mRNA. These data have led to the idea that mRNA is inherently stable in cells and that the rate at which mRNA is degraded is determined by the strength of its destabilizing sequences and not its stabilizing sequences. Of course, this conclusion is based only upon the inability to detect stabilizers and, as a result, serves only as a working model for the field. Several examples of regulated m RNA stability in eukaryotic cells have been reported, and these indicate that the
trans-acting factors of the mRNA decay system can be subject to regulation. For instance, maturation of T cells is in part due to the stimulated decay of specific mRNAs, and this probably results from a cascade of events involving protein kinase C (Takahama and Singer, 1992). Protein kinase C inactivation has also been implicated in the destabilization of several other RNAs (for instance, see Iwai et al., 1991). The regulation of transferrin receptor mRNA stability in response to iron levels is an example of how a destabilizing sequence element can be inactivated by a pathway responding to cellular conditions (Klausner et al., 1993). The cell cycle regulation of histone mRNA levels is an example of how the coordinate synthesis of macromolecules can be controlled by the regulation of mRNA decay (Harris et al., 1991). The stabilization of heat shock protein mRNA at high temperatures highlights the usefulness of stabilizing mRNA only when it is needed (Petersen and Lindquist, 1988). Dramatic changes in the stability of the Ip mRNA in Saccharomyces cerevisiae in response to changes in carbon source suggest the existence of regulated pathways in this organism (Lombardo et al., 1992). Finally, the importance of mRNA decay in differential gene expression is exemplified by the transformation phenotype associated with mRNA stabilization mutations in the c-myc and c-fos mRNAs (reviewed in Schiavi et al., 1992) and the alterations in mRNA-specific destabilizing factors in monocytic tumors (Schuler and Cole,
1988). Specific Mechanisms of Decay Perhaps the most surprising conclusion to come from recent work on mRNA decay is that sequence elements regulating it are found throughout the message (Figure 1). The presence of such a diverse array of elements effectively rules out the simple model that mRNA is passively protected from degradation by nucleases simply by the presence of stabilizing sequences at its ends. The heterogeneous localization of the elements raises the question of whether there can be a common pathway for mRNA decay and how each of the elements could feed into it. Alternatively, if these elements are each acting independently, do they represent nuclease sensitivity sites, or are they recognition sites for other proteins that will eventually recruit a common ribonuclease? Owing to the lack of coherence between studies on many of these regulatory sequences, each of them will be discussed in groups, with each group having in common a specific location on the mRNA.
The 5' Cap Structure Beginning at the 5' end of eukaryotic mRNA is the cap structure. The unique 5'--5' phosphodiester bond of the cap makes it intrinsically resistant to general ribonucleases, and in fact it is this resistance that allowed for its original identification. A specific enzyme that removes the cap (decapping enzyme) has been purified from yeast
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® GpppG
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(9 Figure 1. mRNAStabiitty Is Determined by Many Different Elements The locations of mRNA sequence elements involved in regulating mRNA decay are shown. These are the cap structure (1), 5'UTR secondary structures (2), premature termination codons (3), ORF sequences (4), 3'UTR sequences including the IRE of the transferrin mRNA(5)and the ARE from manyunstablemRNAs(6),andthe poly(A) tail (7).
(Stevens, 1988), and it is possible that an activation of this enzyme by mRNA sequences would create an unstable mRNA. mRNA would be destabilized owing to the presence of a 5'-3'exoribonuclease or, alternatively, an endoribonuclease whose site of action was masked by the cap structure and its associated cap-binding proteins (reviewed in Rhoads, 1993). The existence of a 5'-3' exoribonuclease has been confirmed by the purification and gene cloning of the XRN1 protein in yeast (Larimer et al., 1990) and by the gene cloning of other homologs (Kenna et al., 1993). Complications in the interpretation of the importance of the XRN1 protein in general mRNA decay pathways (Larimer et al., 1992) have arisen since mutations in this protein appear to affect many seemingly unrelated reactions, including nuclear karyogamy, mRNA processing reactions in the nucleus, and recombination (reviewed in Kearsey and Kipling, 1991). Definitive results about the importance of 5'-3' exoribonucleases (and in general about the regulation of cap removal as a mechanism to stimulate mRNA decay) will await a more detailed genetic and biochemical analysis of this family of proteins.
The 5' Untranslated Region and the Translational Requirement of mRNA Degradation The 5' untranslated region (5'UTR) of mRNA has not yet been definitively shown to be a destabilizing sequence on any mRNA. However, 5'UTR sequences have been well documented to control the translatability of an mRNA molecule, and it is through the negative regulation of translation initiation that this region of mRNA has been shown to inhibit the degradation process. An understanding of the relationship between translation initiation and mRNA decay is far from complete, but in general terms it appears to be clear that most mRNAs need to be translated to be degraded and that the requirement for translation may occur at several points in the degradation pathway. Simple experiments in yeast using the translational inhibitor cycloheximide revealed that almost all mRNAs were stabilized by drug treatment (Herrick et al., 1990). In conjunction with this, a mutation resulting in partial loss of function of a transfer RNA (tRNA) nucleotidyl transferase protein, which leads to a decrease in the rates of translation due to limiting functional tRNA, also resulted in mRNA stabilization (Peltz et al., 1992). Although these experiments are well executed, they cannot distinguish between a requirement for translation in mRNA decay and
a requirement for a highly labile degradation activity that fails to be synthesized but continues to be degraded upon the inhibition of translation. Using a different experimental approach, Laso et al. (1993) examined the translatability and steady-state levels of a series of yeast mRNAs differing only in the placement of a stable 5'UTR stem-loop structure. Consistent with earlier experiments by Cigan et al. (1988), these workers find that translation can be inhibited up to 96% by these structures without changing the levels and therefore, by inference, the stability of the mRNA. These data argue very strongly that wild-type rates of translation initiation are not needed for inducing mRNA decay, although they do not address what would happen if mRNA becomes untranslatable. Another experiment addressing the requirement for translation initiation involved studies on the degradation of an mRNA containing the destabilizing AU-rich region (see below) of c-fos (Koeller et al., 1991). In this work, the translation of mRNA was negatively regulated to at least 95% inhibition by the presence of an inhibitory stem, called the iron-responsive element (IRE), within its 5'UTR. Regulation of translation was achieved by altering the levels of iron in the tissue culture media, while the general metabolism and the overall translational capacity of the cell remained unchanged. The data from this study indicated that translation was not required for the rapid degradation of the mRNA. In a very similar experiment, Aharon and Schneider (1993) fused a mutated form of the adenovirus tripartite leader that inhibits translation to a synthetic mRNA construct that was destabilized owing to the presence in its 3'UTR of the AU-rich region of the granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA. In contrast with the results of Koeller et al. (1991), these workers find that the inhibition of translation resulted in the stabilization of the mRNA molecule. In these studies, the relief of translation inhibition and the subsequent appearance of unstable mRNA were achieved by the introduction of an internal ribosome entry site downstream of the 5' inhibitory sequence. In support of these findings, Savant-Bhonsale and Cleveland (1992) find that mRNA containing an AUrich element (ARE) must be translated to be rapidly degraded. Understanding why there are differences between each of these experiments will probably be crucial in comprehending the translation initiation dependence of mRNA decay. For instance, it may be that the AU-rich region of c-fos mRNA has no translational requirement for inducing decay, while the GM-CSF element does. Alternatively, it may be that the efficiency of translational inhibition by the inhibitory 5'UTR elements are different, such that in some cases a limited number of initiation events can occur per mRNA, while in others no initiation occurs (Figure 2). Such a distinction could reveal that the requirement for translation in decay is actually a requirement for recruiting translation factors and that these factors are themselves associated with the degradative enzymes. In support of this model are the findings that a protein needed for translation initiation is also a ribonuclease in yeast (Sachs and Dear-
Review: mRNA Degradation in Eukaryotes 415
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Figure 2. A Minimum Level of Translation Is Required for Efficient mRNA Decay A model describingthe effectsof translationinhibitionon mRNAdecay is shown. Beginningwith a normallyunstablemRNA, the inhibitionof its translation by up to 96% will not affect its rate of turnover. After complete inhibition of the mRNAstranslationby either a stable structure in the 5'UTR or by removalof the initiator methionine,the mRNA becomes stabilized. The lack of a correlation between decay rates and translation initiation efficiency suggests that mRNA only needs to be translatable to be degraded.
dorff, 1992) and that a 20S complex that requires the GMCSF ARE (see below) and whose presence correlates with destabilization by the element requires translation for formation (Savant-Bhonsale and Cleveland, 1992).
Open Reading Frame Destabilizing Sequences Several different experiments have shown that the open reading frame (ORF) region of mRNA contains destabilizing sequences. In what remains a unique example, Cleveland and coworkers discovered that the N-terminal tetrapeptide encoded by the 13-tubulin mRNA provided a signal to target rapid degradation of that mRNA under conditions of tubulin monomer excess (Yen et al., 1988). The molecular mechanism for this induction remains unclear, although physical evidence for it occurring as a cotranslational event has recently been obtained (Theodorakis and Cleveland, 1992). The results of this work highlight the possibility that the ribosome has factors associated with it that can differentially destroy an mRNA molecule and that their activity can be controlled by either mRNA or protein sequence. Experiments utilizing the c-los mRNA also showed the existence of destabilizing sequences within the ORF of the mRNA (Shyu et al., 1989). This mRNA has within its 3'UTR a destabilizing sequence (see below), Shyu et al. (1989) discovered that removal of this 3'UTR element was not sufficient to stabilize the c-fos mRNA (see also Kabnick and Housman, 1988). These authors identified another destabilizing sequence within the ORF and found that it was only utilized when the message was transiently expressed following growth factor stimulation. A closer examination of this element has recently shown that it contains two destabilizing regions, that it is the RNA sequence and not the protein product or a biased codon usage that is required for destabilization, and that translation through the element is required for inducing destabilization (Wellington et al., 1993). This work also shows that the destabilizing determinant appears to induce decay by first stimulating the deadenylation of the mRNA. What remains
mysterious is why the element is only functional when the mRNA is transiently expressed in serum-stimulated cells. More recently, a series of proteins that recognize the ORF destabilizing region of c-fos have been identified by ultraviolet cross-linking experiments (Chen et al., 1992). In a related series of experiments examining an analogous destabilizing sequence in the ORF of c-myc mRNA, Ross and colleagues found that the in vitro decay rates of the polyribosome-bound c-myc mRNA were dramatically increased in the presence of an excess of synthetic RNA containing the destabilizing region (Bernstein et al., 1992). This activation presumably resulted from the competition for a 75 kd protein that recognizes the destabilizing sequence and inactivates it. Both the c-fos and c-myc studies provide some biochemical evidence for the importance of protein complex formation in the regulation of ORF destabilizing determinants. A third example o.f a destabilizing sequence in the ORF is the one found in the yeast MATal mRNA (Parker and Jacobson, 1990). These experiments utilized a yeast strain containing a conditional mutation in the yeast RNA polymerase II subunit (Nonet et al., 1987), thereby allowing a direct measurement of mRNA half-lives without the use of chemical transcription inhibitors. Parker and Jacobson (1990) were able to show that the decay of this mRNA required translation through a 65 nt region. This region contains several rare codons, and more recent experiments by Parker and coworkers (Caponigro et al., 1993) have confirmed that it is the presence of rare codons in conjunction with as yet undefined sequences 3' to them that leads to stimulated decay. These authors hypothesize that the pausing or slowing of ribosomal translocation through this region of the mRNA could expose the RNA to the degradative machinery. Whether or not this machinery is associated with the ribosome remains to be determined. What remains unclear from both these and earlier experiments studying the c-fos ORF destabilizers is whether their requirement for translation is equivalent to or superimposed upon the general requirement for translation in mRNA decay. For instance, besides the putative requirement for translation initiation for all mRNA degradation pathways, the requirements of the elements for elongation through them suggests that elongation activates them, possibly by localizing ribosome-associated degradative enzymes to the destabilizing site. That the requirement for mRNA translation could occur at several different points in the decay reaction hints at the potential complexity of the pathway and justifies a more detailed analysis of the relationship between decay and translation. Furthermore, distinguishing between the general and specific requirements for translation in mRNA decay will be essential in discerning the mode of action of different destabilizing elements.
Premature Termination and mRNA Destabilization Investigations on the mechanism by which nonsense mutations within mRNA lead to instability have provided significant insight into the general pathways of mRNA decay. Although it had been appreciated for some time that non-
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UPF1
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Figure 3. The Degradationof mRNA ContainingPrematureTermination Codons in Yeast Requiresthe UPF1 Protein and Other mRNA Sequence Elements Deletion of the UPF1 gene (UPFIA) results in the stabihzationof mRNAs containing premature termination codons without affecting the rates of normal mRNA decay. Destabilizationby the termination codonsappearsto requirea downstreamsequencecontaininga methionine codon and the absence of an as yet ill-defined stabilizer sequence(X)just upstream.See Peltzet al. (1993)andthe textfor details.
sense mutations can destabilize mRNA and that their ability to do so decreases as they move further away from the initiator methionine of the message (Losson and Lacroute, 1979), a detailed description of the process had not been achieved. Work on allosuppressors of nonsense mutations in the yeast HIS4 mRNA changed this when it became clear that one of these suppressors, upfl, functioned by stabilizing nonsense-containing messages (Leeds et al., 1991). Studies on other allosuppressors identified in this screen have recently revealed that they also work by stabilizing the mRNA (Leeds et al., 1992). The UPF1 gene product can be deleted from yeast with no alterations in viability on normal medium (Altamura et al., 1992; Leeds et al., 1992). In UPFl-deleted cells, nonsense mRNAs are stabilized back to the levels of their wild-type counterpart (Figure 3). Furthermore, the pathways for general mRNA decay remain unaffected. These data argue that the UPF1 pathway in yeast is distinct from the general mRNA decay pathway and that it is dispensable. However, like the general pathway, nonsensemediated decay requires translation. The stabilization of intron-containing mRNAs in the cytoplasm of upfl mutant cells indicates that besides destroying mutated mRNA, this pathway could be responsible for removing incompletely processed mRNA that may escape retention controls within the nucleus (He et al., 1993). An explanation for why nonsense mutations exhibit a loss of destabilizing activity as they are moved further away from the initiator methionine has recently been presented (Figure 3). By utilizing either wild-type or mutant UPF1 strains, Peltz et al. (1993) were able to show that this distance dependence was created by several distinct RNA sequence elements. First, as expected, the presence of a nonsense mutation was essential for UPFl-mediated decay. Second, the presence of a downstream sequence was also required, and point m utagenesis of this sequence suggested methionine codons were an essential part of it. Finally, the UPF1 pathway was inactivated by the presence of a protection sequence preceding the nonsense mutation. These data are consistent with the model that the transiting ribosome can be made insensitive to nonsense mutations and that sensitivity only occurs when the ribosome
has the ability to reinitiate downstream of the nonsense mutation. Nonsense mutations would therefore lose their destabilizing activity as they move further away from the initiator codon because of the increase in probability of being 3' to a protection sequence and not 5' to another initiator codon. Because the protection sequences are normally found on the RNA, it is probable that they serve another function. One possible role for these sequences would be to prepare the ribosome for termination. If mRNA contains such termination preparation sequences, then it may turn out that the position of ORF destabilizing sequences relative to this sequence may determine how efficiently they control the stability of the message.
3'UTR Destabilizing Sequences Sequences that regulate stability within the 3'UTR of mRNA have received the greatest attention in the mRNA degradation field. The two most well-studied examples are the IRE on the transferrin mRNA and the ARE found on many unstable mRNAs. Taken together, investigations into the mode of action of these two elements have revealed the depth of regulation that probably impinges upon other less well-characterized elements. The regulation of transferrin mRNA degradation in response to changes in cellular stores of iron has been the subject of a recent review (Klausner et al., 1993), so only a brief synopsis of the important findings will be discussed here. Located within the 3'UTR of this mRNA is a region that contains five distinct stem-loop structures capable of binding the IRE-binding protein (IRE-BP). The affinity of this protein for these sequences is what is regulated by cellular iron, and these changes in affinity occur through dissociation and reassociation of an iron-sulfer cluster within the IRE-BP. Interestingly, the IRE-BP also possesses aconitase activity, suggesting an unusual but possibly important linkage between general cellular metabolism and RNA metabolism. The binding of the IRE-BP to the transferrin mRNA stabilizes the mRNA. The mRNA itself is not destabilized owing to the IRE structures, but is instead made unstable owing to the presence of an as yet uncharacterized destabilizing sequence in the vicinity of the stern-loop structures. The current model for how the IRE-BP stabilizes mRNA is that binding to the IREs prevents association of destabilizing factors to the destabilizing sequence. Whether these destabilizing factors are themselves ribonucleases or whether they recruit ribonucleases to destroy the mRNA at a distinct site remains unclear. The regulation of transferrin mRNA decay provides at least two important mechanistic insights. First, it shows how the decay process can be regulated by proteins that can directly respond to changes in the cellular environment. Second, the transferrin mRNA studies reveal that mRNA stabilization may in fact result from an inhibition of an activity recognizing the destabilizing element and that this destabilizing element can be distant from the binding site of the stabilizing proteins. A similar inhibition of mRNA destabilizing sequences by distant sequence elements has been described by Heaton et al. (1992). In this example, a 3'UTR destabilizing
Rewew: mRNA Degradation in Eukaryotes 417
sequence from the yeast STE3 mRNA was found to stimulate degradation of the normally stable yeast PGK1 mRNA only when a portion of the PGK10RF had been removed. The inability of a destabilizing sequence to work in certain contexts raises the possibility that the interaction between different regions of mRNA can lead to increases or decreases in the efficiency of element utilization. Such regulation in cis by DNA sequence elements surrounding transcriptional promoters is well documented, and these data provide some evidence that mRNA metabolism reactions will be subject to similar hierarchical controls. The second well-characterized 3'UTR destabilizing sequence is the ARE. The original observation by Shaw and Kamen (1986) that an ARE in the 3'UTR region of the G M CSF mRNA could stimulate the degradation of the stable 13-giobin mRNA provided much of the basis for current work in identifying destabilizing sequences in general and for understanding the mechanism of AU-rich-stimulated decay in particular. The sequence consensus for the AREs is loosely defined as AUUUA repeated once or several times within the 3'UTR. This pentanucleotide is often found within a U-rich region of the mRNA. Characteristic classes of ARE-containing cellular mRNAs include the lymphokine genes and the immediate early genes. The absence of a clear understanding of the key sequence features of the ARE that allow it to function has precluded a detailed characterization of the involvement of sequences surrounding it. Because of this, the presence of an ARE in a 3'UTR is not sufficient to deem it a destabilizing sequence since it could be inactivated by neighboring sequences. Furthermore, not all AREs are the same. For instance, it has been shown that only a subset of AREcontaining mRNAs are stabilized following changes in cellular metabolism (Lindstein et al., 1989; Schuler and Cole, 1988). A large number of laboratories have examined the proteins bound to the ARE of various mRNAs (Bickel et al., 1992; Bohjanen et al., 1991, 1992; Brewer, 1991; Gillis and Malter, 1991; Hamilton et al., 1993; Savant-Bhonsale and Cleveland, 1992; Vakalopoulou et al., 1991; You et al., 1992). The different methodologies include in vivo or in vitro ultraviolet cross-linking, partial fractionation based upon stimulated decay in in vitro systems, or examination of the differences in sedimentation velocities of RNAs containing AREs and AREs harboring inactivating point mutations. The details of these experiments are too complex to be described here, although several general conclusions can be made. First, it is clear that both nuclear and cytoplasmic proteins can specifically bind to the ARE. The significance of the nuclear-binding proteins remains obscured by the lack of knowledge concerning the contribution of nuclear mRNA processing reactions to the ultimate fate of mRNA in the cytoplasm. Second, the binding activity of some of these proteins appears to increase or decrease in response to the changes in cellular metabolism that lead to alterations in the stabilities of ARE-containing mRNAs. Finally, the existence of a 20S complex on an unstable ARE-containing mRNA and the absence of this complex on ARE-containing mRNAs that are stable owing to ARE inactivation (Savant-Bhonsale and Cleveland,
1992) suggest that ARE activity could be mediated by a complex of proteins (Figure 4). The studies on the ARE-bound proteins are incomplete for several reasons. First, there appear to be two classes of binding proteins associating with the ARE. One class binds equally well to poly(U), while the other appears to be specific for certain AREs. Which class contains proteins that mediate ARE function is not clear. The studies are also incomplete because the relative abundances of each of these proteins remains unknown. For instance, recent data (Hamilton et al., 1993) suggest that many of these proteins may be members of the well-characterized abundant class of heterogeneous nuclear RNA-binding proteins. If true, this would raise serious concerns about the importance of these proteins in destabilization since their cytoplasmic localization has only been detected under conditions of transcriptional arrest (PiSoI-Roma and Dreyfuss, 1992). Because relative abundances are not known, it is unclear whether the cross-linking experiments are detecting specific regulatory proteins or whether general m RNA-binding proteins are showing preferences for either poly(U) or certain AREs. Finally, the studies are incomplete because the absence of an in vitro system in all but one of these studies (Brewer, 1991) results in conclusions based on correlations between changes in cross-linking activity of a protein and measurable changes in mRNA half-lives. Furthermore, no direct confirmation that these proteins mediate ARE function in vivo has been reported. Because of these problems, this group of ARE-binding proteins can only tentatively be considered to be key players in ARE function. Although the role of the ARE-binding proteins remains unclear, the mechanism by which this element stimulates decay has been elucidated in broad outline. Shyu et al. (1991) have convincingly shown that the ARE from the c-fos mRNA mediates decay by first stimulating deadenylation and then providing an element that stimulates the next phase of the degradation process. Uncoupling of the two components of the ARE-mediated response was achieved by point mutations that maintained the ability of
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Figure 4. 20S Complex Formationon mRNAContainingan ARE Occurs Only When the Element Is Active The abmlityof the ARE to stimulate mRNA decay requires that the message be translated, that the ARE not be translated, and that the termination of translationoccurs 5' to the ARE. These requirements can be interpretedto reflect the conditions under which a large 20S particle is formed and maintainedon the mRNA.This particleis potentially the protein complex that mediates the destabilizing properties of the elements. See Savant-Bhonsaleand Cleveland(1992) and the text for details.
Cell 418
the ARE to stimulate deadenylation but inhibited its ability to stimulate mRNA decay. Several other ARE-containing mRNAs have been found that go through a deadenylation and then decay pathway using similar techniques (LairdOffringa et al., 1990; Stoeckle, 1992; Wilson and Treisman, 1988). Together with the work on c-los, these data strongly argue that deadenylation may be a prerequisite for the decay of this class of unstable mRNAs and that deadenylated mRNA is an early intermediate in the degradation process.
Poly(A) Tail Removal and mRNA Degradation How general is the sequential pathway of deadenylation followed by mRNA degradation? Some of the earliest proposals on poly(A) tail function were that the tail provided a buffer to the mRNA from exonucleases and that the pathway of degradation would involve an obligate deadenylation step followed by a continuation of the exonucleolytic degradation in the 3' to 5' direction (reviewed in Peltz et al., 1991). Subsequent experiments investigating the sequential nature of decay reactions for several mRNAs provided ambiguous data. The recent advances in technologies that allow the precise measurement of short poly(A) tails by utilizing RNAase H-directed cleavage of mRNA and the availability of inducible and repressible promoters in several systems (for instance, see Helms and Rottman, 1990; Muhlrad and Parker, 1992; Shyu et al., 1989) have allowed a reexamination of whether mRNA must be deadenylated before it is degraded. A systematic study of the timing of deadenylation relative to the decay of several yeast mRNAs utilizing the yeast activatable and repressible galactose promoter has revealed that the mRNAs are deadenylated before they are degraded (Decker and Parker, 1993). These data are consistent with the effects of two conditional mutations in yeast that lead to an enhancement of both poly(A) and mRNA destabilization (Minvielle-Sebastia et al., 1991). They are also consistent with the data of Wellington et al. (1993) showing that ORF destabilizers induce deadenylation. The experiments by Decker and Parker (1993) are surprising since the mRNAs examined exhibit the full range of stability in the cell. As a result, they suggest that the pathway of deadenylation preceding decay may not be limited to just the highly unstable messages. These data are an extension of work studying the degradation pathway of the yeast mating factor ~2 (MFA2) mRNA (Muhlrad and Parker, 1992). In this work, a destabilizing sequence within the 3'UTR of the message was found to behave in a fashion analogous to the ARE from higher cells in that it stimulated the degradation of the mRNA by stimulating its deadenylation. Importantly, mutations that inhibited the rates of MFA2 deadenylation also decreased the rates of mRNA decay. This work highlighted the importance of distinguishing between the shortening of poly(A) tails down to approximately 12-15 residues and the shortening of these oligoadenylate tails to lengths below 10 nt in a process called terminal deadenylation (see below). The recent study by Decker and Parker (1993) also iden-
titles an in vivo cleaved mRNA degradation intermediate that shows temporally correct precursor-product relationships. Previous studies had only detected intermediates in a steady-state population of mRNA (Binder et al., 1989; Lim and Maquat, 1992; Stoeckle, 1992; Stoeckle and Hanafusa, 1989; Vreken and Rau~, 1992). This degradation intermediate was stabilized by a 5' tract of G residues, which had been shown previously to protect mRNA fragments from 5' exonucleases (Vreken and Rau~, 1992). The intermediate was a fragment of mRNA containing the 3' end of the molecule with several adenines at the tail. The existence of this intermediate in vivo shows that although deadenylation is a prerequisite for initiating mRNA decay, the subsequent nucleolytic reactions can occur within the message.
Pathways of mRNA Degradation and Trans-Acting Factors Using the data summarized in this review, a general model for mRNA degradation can be created (Figure 5). mRNAs are not degraded until their tails are shortened to lengths below 10 nt, a length that is incapable of high affinity binding to the poly(A)-binding protein (see below). The regulation of mRNA decay can result from alterations in the rates of poly(A) tail shortening or terminal deadenylation since poly(A) removal appears to be rate limiting in the decay process. Once deadenylation is completed, there is a rapid degradation of the mRNA that occurs by either endo- or exonucleases. The coupling of the activation of these nucleases, which remain undefined, to the completion of deadenylation explains why it is so difficult to find intact but terminally deadenylated mRNA within the cell.
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Figure 5. DeadenylationMay Be a Prerequisitefor mRNA Degradation Processiveshorteningof poly(A)tails by the normallydistributivepoly(A) nucleasecould be inducedby mRNAdestabilizingsequences. The rate of terminaldeadenylatloncouldalso be regulatedby these elements. Oncedeadenylated,the mRNAcan be destroyedby many differenttypesof ribonucleases.Theactivationof the degradationstep is tightlycoupledto the completionof deadenylation,and as a result alterations in deadenylationrates will changemRNA half-lives.See the text for details.
Review:mRNADegradationin Eukaryotes 419
The degradation pathway of mRNAs containing a premature termination codon is probably different than that for normal mRNAs. One indication of this is the lack of requirement for the UPF1 pathway for yeast cell viability and the lack of effect of the UPF1 deletion on normal mRNA decay rates. Furthermore, Shyu et al. (1991) have found that I~-globin mRNA containing a destabilizing nonsense mutation is degraded before it is deadenylated. The mechanism of action of destabilizing sequences within mRNA can be rationalized from these data. These sequences require translation for activitysince their recognition is mediated by factors brought to the mRNA by the translational machinery. Once recognized, the elements induce decay by activating poly(A) tail removal. Following the terminal deadenylation step, identical or other sequence elements within mRNA are recognized by the exoor endonucleases that eventually lead to the destruction of the mRNA. Several important predictions can be made from this model. First, if deadenylation is a common initial step in the degradation pathway of mRNAs, then the target of each of the destabilizing sequences may be to activate the single event of deadenylation. As a result, an understanding of destabilizing sequence activity will come through examining how they activate this process. Second, this model predicts that the identification of decay intermediates in vivo or in in vitro reactions will not necessarily give insight into what the rate-limiting step in the decay process is. For instance, endonucleolytic cleavage of deadenylated mRNA may be much faster than deadenylation, and as a result the intact deadenylated intermediate will not be seen owing to its rapid metabolism. Finally, this model predicts that elements within an mRNA can effect degradation events occurring elsewhere on the mRNA. If deadenylation is a key step in the regulation of mRNA stability, then an understanding of the biochemistry of this step will be essential for a dissection of decay pathways. mRNA polyadenylation occurs in the nucleus of cells, and the posttranscriptionally synthesized tail is typically homogeneous in length, ranging from 70-90 nt in yeast to 220250 nt in mammalian cells (reviewed in Sachs, 1990). Following transport of the mRNA, the long poly(A) tail is shortened in the cytoplasm. In yeast, this shortening reaction requires the presence of a specific protein bound to the poly(A) tail, the poly(A)-binding protein, for full efficiency (Sachs and Davis, 1989), and it is this reaction that could be regulated by the different destabilizing sequences on many RNAs. As described above, the poly(A) shortening reaction can be separated into two phases, the first being the shortening of the tail down to 12-25 residues and the second terminal deadenylation step being the removal of some or all of these residues. The ribonuclease involved in poly(A)-binding proteindependent shortening of poly(A) tails from yeast has been purified and cloned (Sachs and Deardorft, 1992). This poly(A) ribonuclease (PAN) is unique among identified eukaryotic RNAases in that it requires a protein-RNA complex as a substrate. Conditional mutations in PAN reveal
it is also a translation initiation factor. PAN deadenylation activity in vitro exhibits the two phases of shortening seen in vivo, and both the shortening phase and the terminal deadenylation phase can be regulated by 3'UTR sequences in a reconstituted system containing highly purified enzyme, poly(A)-binding protein, and synthetic RNA (Lowell et al., 1992). In this system, the stimulation of PAN shortening activity by sequence elements within the 3'UTR of the yeast MFA2 mRNA occurs by switching PAN from a distributive to a highly processive enzyme. The mechanism by which this switch occurs is under investigation, but it is probably analogous to the mechanism by which AREs (and potentially other destabilizing sequences within the mRNA) stimulate deadenylation of the poly(A) tail. The inhibition of the terminal deadenylation step by a sequence element within the 3'UTR of a synthetic mRNA in the in vitro system suggests that poly(A) tail stabilization by sequence elements within stable messages could also be occurring in vivo. The characterization of PAN reveals some interesting properties that may become more general for RNA degradation reactions. First, the substrate for the ribonuclease is not naked mRNA, suggesting that the search for RNAases that specifically recognize sequences within mRNA that have been identified as destabilizing sequences may be unsuccessful unless the proper ribonucleoprotein substrate is identified. That the nucleolytic substrate could be a ribonucleoprotein complex negates the simple hypothesis that RNA-binding proteins will always protect mRNA from decay. Second, PAN activity can be regulated from distant sites. This argues that destabilizing sequences may not harbor nucleolytic sites, but instead target sites elsewhere on the mRNA for degradation. A corollary of this is that the identification of endonucleolytic cleavage sites on RNA decay intermediates may not yield information about what region of the mRNA made these sites nuclease sensitive. Furthermore, the ability of PAN to be activated or inhibited over distances in vitro is consistent with the hypothesis that destabilizing sequences located throughout an mRNA molecule can affect the deadenylation and decay pathways. Last, the requirement for PAN in both translation and (presumably) RNA decay in vivo leads to the suggestion that some of the factors involved in mRNA decay may also be complexed with translation initiation factors. This conclusion is consistent with the apparent requirement for translation in mRNA decay discussed above. Other in vitro systems have also been utilized to explore mRNA decay pathways, but none have utilized pure components. The most successful in vitro reactions appear to be those that examine the degradation of mRNA attached to polyribosomes (Brewer, 1991; Vreken et al., 1992). Interestingly, the mRNAs in these experiments appear to be degraded, even though the polyribosomes are incubated under conditions that do not allow translation. The significance of this may only become clear after further work examining the in vivo requirements for translation. Finally, a mammalian poly(A)-specific nuclease has been partially purified (,~,strom et al., 1992), although the relationship of
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this enzyme to the yeast PAN and its importance in mRNA decay reactions are not yet known.
Summary and Perspectives mRNA contains within it the information needed to determine its stability within the cell. Destabilizing information lies within discrete regions of sequence, and these sequence elements can be found throughout the message and may be redundant. Many destabilizing elements require translation for activity, although the reasons for this are not understood. The masking or activation of these elements by other proteins probably leads to regulated mRNA decay rates. The target of these elements could be the deadenylation machinery since many mRNAs are only degraded after the rate-limiting step of deadenylation is completed. Once the mRNA is deadenylated, these or other elements may provide nucleolytic sites that allow for the rapid degradation of the mRNA. A more complete understanding of mRNA decay will in part require more detailed descriptions of the sequences within m RNAs that determine instability. Equally important will be a thorough in vivo analysis of how an mRNA is degraded, an understanding of why there is a requirement for translation, and the creation of in vitro RNA decay systems that accurately reflect the in vivo decay pathways. Current work in the field utilizing a combination of biochemical and genetic approaches will give insight into each of these areas, and with this should come the discovery and subsequent elucidation of the regulatory networks impinging on these processes.
Acknowledgments I thank Allan Jacobson and Matthew Sachs for critical reading of the review and each of the laboratories that provided preprints of their manuscripts. Work in the laboratory of the author is supported by National Institutes of Health grant R29 GM-43164 and in part by awards from the Markey, Searle Scholars, and March of Dimes Foundations.
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of mRNA. Curr. Opin. Cell Biol. 2, 1092-1098. Sachs, A. B., and Davis, R. W. (1989). The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell 58, 857-867. Sachs, A. B., and Deardorff, J. A. (1992). Translation inttiation requires the PAB-dependent poly(A) ribonuclease in yeast. Cell 70, 961-973. Savant-Bhonsale, S., and Cleveland, D. W. (1992). Evidence for mstability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a >20S degradation complex. Genes Dev. 6, 1927-1939. Schiavi, S. C., Belasco, J. G., and Greenberg, M. E. (1992). Regulation of proto-oncogene mRNA stability. Biochim. Biophys. Acta 1114, 95106. Schuler, G. D., and Cole, M. D. (1988). GM-CSF and oncogene mRNA stabilities are independently regulated in trans in a mouse monocytic tumor. Cell 55, 1115-1122. Shaw, G., and Kamen, R. (1986). A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation Cell 46, 659-667. Shyu, A.-B., Greenberg, M. E., and Belasco, J. G. (1989). The c-fos transcript is targeted for rapid decay by two distinct m RNA degradation pathways. Genes Dev. 3, 60-72. Shyu, A.-B., Belasco, J. G., and Greenberg, M. E. (1991). Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes Dev. 5, 221-231. Stevens, A. (1988). mRNA-decapping enzyme from Saccharomyces cerevisiae: purification and unique substrate specificity for long RNA chains. Mol Cell. Biol. 8, 2005-2010. Stoeckle, M. Y. (1992). Removal of a 3' non-coding sequence is an initial step in the degradation of groa mRNA and is regulated by interleukin-l. Nucl. Acids Res. 20, 1123-1127. Stoeckle, M. Y., and Hanafusa, H. (1989). Processmg of 9E3 mRNA and regulation of its stability in normal and Rous sarcoma virustransformed cells. Mol. Cell. Biol. 9, 4738-4745. Takahama, Y., and Singer, A. (1992). Post-transcriptional regulation of early T cell development by T cell receptor signals. Science 258, 1456-1462. Theodorakis, N. G., and Cleveland, D. W. (1992). Physical evidence for cotranslational regulation of ~-tubulin mRNA degradatton. Mol. Cell. Biol. 12, 791-799. Vakalopoulou, E., Schaack, J., and Shenk, T. (1991). A 32-kilodalton protein binds to AU-rich domains in the 3' untranslated regions of rapidly degraded mRNAs. Mol. Cell. Biol. 11, 3355-3364. Vreken, P., and Rau6, H. A. (1992). The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region. Mol. Cell. Biol. 12, 2986-2996. Vreken, P., Buddelmeijer, N., and Rau~, H. A. (1992). A cell-free extract from yeast cells for studying mRNA turnover. Nucl. Acids Res. 20, 2503-2510. Wellington, C. L., Greenberg, M. E., and Belasco, J. G. (1993). The destabilizing elements in the coding region of c-fos mRNA are recognized as RNA. Mol. Cell. Biol. 13, in press. Wilson, T., and Treisman, R. (1988). Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3' AU-rich sequences. Nature 336, 396-399. Yen, T. J., Gay, D. A., Pachter, J. S., and Cleveland, D. W. (1988). Autoregulated changes in stability of polyribosome-bound J3-tubulin mRNAs are specified by the first thirteen translated nucleotides. Mol. Cell. Biol. 8, 1224-1235. You, Y., Chen, C.-Y. A., and Shyu, A.-B. (1992). U-rich sequencebinding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation. Mol. Cell. Biol. 12, 29312940.
Review
Messenger RNA Degradation in Eukaryotes Alan B, Sachs Division of Biochemistry and Molecular Biology University of California, Berkeley Berkeley, California 94720
Introduction Messenger RNA (mRNA) degradation in eukaryotic cells is a regulated process that can determine the level of expression of a gene. In particular, the existence of highly unstable RNAs allows for rapid and precise reductions or elevations in transcript levels following changes in transcript production rates or alterations in the machinery dedicated to transcript degradation. The mechanisms by which mRNAs are degraded and the sequence elements within the mRNAs that determine their stability are the subject of this review. A more detailed treatment of this topic and of prokaryotic mRNA degradation can be found in a recent collection of reviews (Belasco and Brawerman, 1993). The range of mRNA stability in eukaryotic cells can vary over several orders of magnitude (reviewed in Peltz et al., 1991). For instance, in higher cells some mRNAs are degraded with a half-life of about 20 min, while in the same cell, other mRNAs are degraded with half-lives over 24 hr. Similar differences in mRNA half-lives have been found in yeast, with the most unstable messages decaying in less than 5 min and the most stable decaying at rates slower than 60 min (Herrick et al., 1990). Searches for sequence elements on mRNA that modulate decay rates have revealed several types of destabilizing elements (see below). These elements are identified by their ability to confer destabilization to a normally stable message. Interestingly, removal of these sequences from their native context often does not lead to stabilization of that message. This has been interpreted to suggest that mRNAs have multiple destabilizing sequences and that the process by which mRNA is targeted to decay may be redundant within the message. The search for mRNA stabilizing sequences has been much less successful. In some cases, sequence elements that silence destabilizing sequences have been found (see below), but in no case has a sequence by itself been found to confer stability to another mRNA. The exceptions to this are regions of RNA secondary structure that can stabilize mRNA fragments produced during the decay reaction (Decker and Parker, 1993; Vreken and Rau6, 1992) and the cap and poly(A) tail of the mRNA. These data have led to the idea that mRNA is inherently stable in cells and that the rate at which mRNA is degraded is determined by the strength of its destabilizing sequences and not its stabilizing sequences. Of course, this conclusion is based only upon the inability to detect stabilizers and, as a result, serves only as a working model for the field. Several examples of regulated m RNA stability in eukaryotic cells have been reported, and these indicate that the
trans-acting factors of the mRNA decay system can be subject to regulation. For instance, maturation of T cells is in part due to the stimulated decay of specific mRNAs, and this probably results from a cascade of events involving protein kinase C (Takahama and Singer, 1992). Protein kinase C inactivation has also been implicated in the destabilization of several other RNAs (for instance, see Iwai et al., 1991). The regulation of transferrin receptor mRNA stability in response to iron levels is an example of how a destabilizing sequence element can be inactivated by a pathway responding to cellular conditions (Klausner et al., 1993). The cell cycle regulation of histone mRNA levels is an example of how the coordinate synthesis of macromolecules can be controlled by the regulation of mRNA decay (Harris et al., 1991). The stabilization of heat shock protein mRNA at high temperatures highlights the usefulness of stabilizing mRNA only when it is needed (Petersen and Lindquist, 1988). Dramatic changes in the stability of the Ip mRNA in Saccharomyces cerevisiae in response to changes in carbon source suggest the existence of regulated pathways in this organism (Lombardo et al., 1992). Finally, the importance of mRNA decay in differential gene expression is exemplified by the transformation phenotype associated with mRNA stabilization mutations in the c-myc and c-fos mRNAs (reviewed in Schiavi et al., 1992) and the alterations in mRNA-specific destabilizing factors in monocytic tumors (Schuler and Cole,
1988). Specific Mechanisms of Decay Perhaps the most surprising conclusion to come from recent work on mRNA decay is that sequence elements regulating it are found throughout the message (Figure 1). The presence of such a diverse array of elements effectively rules out the simple model that mRNA is passively protected from degradation by nucleases simply by the presence of stabilizing sequences at its ends. The heterogeneous localization of the elements raises the question of whether there can be a common pathway for mRNA decay and how each of the elements could feed into it. Alternatively, if these elements are each acting independently, do they represent nuclease sensitivity sites, or are they recognition sites for other proteins that will eventually recruit a common ribonuclease? Owing to the lack of coherence between studies on many of these regulatory sequences, each of them will be discussed in groups, with each group having in common a specific location on the mRNA.
The 5' Cap Structure Beginning at the 5' end of eukaryotic mRNA is the cap structure. The unique 5'--5' phosphodiester bond of the cap makes it intrinsically resistant to general ribonucleases, and in fact it is this resistance that allowed for its original identification. A specific enzyme that removes the cap (decapping enzyme) has been purified from yeast
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® GpppG
® AUG
UAA
(NNNN) UAA
(A
A)n (AAA)n
(9 Figure 1. mRNAStabiitty Is Determined by Many Different Elements The locations of mRNA sequence elements involved in regulating mRNA decay are shown. These are the cap structure (1), 5'UTR secondary structures (2), premature termination codons (3), ORF sequences (4), 3'UTR sequences including the IRE of the transferrin mRNA(5)and the ARE from manyunstablemRNAs(6),andthe poly(A) tail (7).
(Stevens, 1988), and it is possible that an activation of this enzyme by mRNA sequences would create an unstable mRNA. mRNA would be destabilized owing to the presence of a 5'-3'exoribonuclease or, alternatively, an endoribonuclease whose site of action was masked by the cap structure and its associated cap-binding proteins (reviewed in Rhoads, 1993). The existence of a 5'-3' exoribonuclease has been confirmed by the purification and gene cloning of the XRN1 protein in yeast (Larimer et al., 1990) and by the gene cloning of other homologs (Kenna et al., 1993). Complications in the interpretation of the importance of the XRN1 protein in general mRNA decay pathways (Larimer et al., 1992) have arisen since mutations in this protein appear to affect many seemingly unrelated reactions, including nuclear karyogamy, mRNA processing reactions in the nucleus, and recombination (reviewed in Kearsey and Kipling, 1991). Definitive results about the importance of 5'-3' exoribonucleases (and in general about the regulation of cap removal as a mechanism to stimulate mRNA decay) will await a more detailed genetic and biochemical analysis of this family of proteins.
The 5' Untranslated Region and the Translational Requirement of mRNA Degradation The 5' untranslated region (5'UTR) of mRNA has not yet been definitively shown to be a destabilizing sequence on any mRNA. However, 5'UTR sequences have been well documented to control the translatability of an mRNA molecule, and it is through the negative regulation of translation initiation that this region of mRNA has been shown to inhibit the degradation process. An understanding of the relationship between translation initiation and mRNA decay is far from complete, but in general terms it appears to be clear that most mRNAs need to be translated to be degraded and that the requirement for translation may occur at several points in the degradation pathway. Simple experiments in yeast using the translational inhibitor cycloheximide revealed that almost all mRNAs were stabilized by drug treatment (Herrick et al., 1990). In conjunction with this, a mutation resulting in partial loss of function of a transfer RNA (tRNA) nucleotidyl transferase protein, which leads to a decrease in the rates of translation due to limiting functional tRNA, also resulted in mRNA stabilization (Peltz et al., 1992). Although these experiments are well executed, they cannot distinguish between a requirement for translation in mRNA decay and
a requirement for a highly labile degradation activity that fails to be synthesized but continues to be degraded upon the inhibition of translation. Using a different experimental approach, Laso et al. (1993) examined the translatability and steady-state levels of a series of yeast mRNAs differing only in the placement of a stable 5'UTR stem-loop structure. Consistent with earlier experiments by Cigan et al. (1988), these workers find that translation can be inhibited up to 96% by these structures without changing the levels and therefore, by inference, the stability of the mRNA. These data argue very strongly that wild-type rates of translation initiation are not needed for inducing mRNA decay, although they do not address what would happen if mRNA becomes untranslatable. Another experiment addressing the requirement for translation initiation involved studies on the degradation of an mRNA containing the destabilizing AU-rich region (see below) of c-fos (Koeller et al., 1991). In this work, the translation of mRNA was negatively regulated to at least 95% inhibition by the presence of an inhibitory stem, called the iron-responsive element (IRE), within its 5'UTR. Regulation of translation was achieved by altering the levels of iron in the tissue culture media, while the general metabolism and the overall translational capacity of the cell remained unchanged. The data from this study indicated that translation was not required for the rapid degradation of the mRNA. In a very similar experiment, Aharon and Schneider (1993) fused a mutated form of the adenovirus tripartite leader that inhibits translation to a synthetic mRNA construct that was destabilized owing to the presence in its 3'UTR of the AU-rich region of the granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA. In contrast with the results of Koeller et al. (1991), these workers find that the inhibition of translation resulted in the stabilization of the mRNA molecule. In these studies, the relief of translation inhibition and the subsequent appearance of unstable mRNA were achieved by the introduction of an internal ribosome entry site downstream of the 5' inhibitory sequence. In support of these findings, Savant-Bhonsale and Cleveland (1992) find that mRNA containing an AUrich element (ARE) must be translated to be rapidly degraded. Understanding why there are differences between each of these experiments will probably be crucial in comprehending the translation initiation dependence of mRNA decay. For instance, it may be that the AU-rich region of c-fos mRNA has no translational requirement for inducing decay, while the GM-CSF element does. Alternatively, it may be that the efficiency of translational inhibition by the inhibitory 5'UTR elements are different, such that in some cases a limited number of initiation events can occur per mRNA, while in others no initiation occurs (Figure 2). Such a distinction could reveal that the requirement for translation in decay is actually a requirement for recruiting translation factors and that these factors are themselves associated with the degradative enzymes. In support of this model are the findings that a protein needed for translation initiation is also a ribonuclease in yeast (Sachs and Dear-
Review: mRNA Degradation in Eukaryotes 415
Stablhtv GpppG
~
GpppG - - ~
GpppG~ 1~
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UAG
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~ ~ ~ UAA C._X_~C._~(..~
r) AUG
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~
Reference
~AAAIn
unstable
UAA
(AAA)n
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UAA
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stable
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UAA ]n'~IJ--'~'~'-I~(AAA)n
Laso et al (1993) K~ller et al (1991)
Figure 2. A Minimum Level of Translation Is Required for Efficient mRNA Decay A model describingthe effectsof translationinhibitionon mRNAdecay is shown. Beginningwith a normallyunstablemRNA, the inhibitionof its translation by up to 96% will not affect its rate of turnover. After complete inhibition of the mRNAstranslationby either a stable structure in the 5'UTR or by removalof the initiator methionine,the mRNA becomes stabilized. The lack of a correlation between decay rates and translation initiation efficiency suggests that mRNA only needs to be translatable to be degraded.
dorff, 1992) and that a 20S complex that requires the GMCSF ARE (see below) and whose presence correlates with destabilization by the element requires translation for formation (Savant-Bhonsale and Cleveland, 1992).
Open Reading Frame Destabilizing Sequences Several different experiments have shown that the open reading frame (ORF) region of mRNA contains destabilizing sequences. In what remains a unique example, Cleveland and coworkers discovered that the N-terminal tetrapeptide encoded by the 13-tubulin mRNA provided a signal to target rapid degradation of that mRNA under conditions of tubulin monomer excess (Yen et al., 1988). The molecular mechanism for this induction remains unclear, although physical evidence for it occurring as a cotranslational event has recently been obtained (Theodorakis and Cleveland, 1992). The results of this work highlight the possibility that the ribosome has factors associated with it that can differentially destroy an mRNA molecule and that their activity can be controlled by either mRNA or protein sequence. Experiments utilizing the c-los mRNA also showed the existence of destabilizing sequences within the ORF of the mRNA (Shyu et al., 1989). This mRNA has within its 3'UTR a destabilizing sequence (see below), Shyu et al. (1989) discovered that removal of this 3'UTR element was not sufficient to stabilize the c-fos mRNA (see also Kabnick and Housman, 1988). These authors identified another destabilizing sequence within the ORF and found that it was only utilized when the message was transiently expressed following growth factor stimulation. A closer examination of this element has recently shown that it contains two destabilizing regions, that it is the RNA sequence and not the protein product or a biased codon usage that is required for destabilization, and that translation through the element is required for inducing destabilization (Wellington et al., 1993). This work also shows that the destabilizing determinant appears to induce decay by first stimulating the deadenylation of the mRNA. What remains
mysterious is why the element is only functional when the mRNA is transiently expressed in serum-stimulated cells. More recently, a series of proteins that recognize the ORF destabilizing region of c-fos have been identified by ultraviolet cross-linking experiments (Chen et al., 1992). In a related series of experiments examining an analogous destabilizing sequence in the ORF of c-myc mRNA, Ross and colleagues found that the in vitro decay rates of the polyribosome-bound c-myc mRNA were dramatically increased in the presence of an excess of synthetic RNA containing the destabilizing region (Bernstein et al., 1992). This activation presumably resulted from the competition for a 75 kd protein that recognizes the destabilizing sequence and inactivates it. Both the c-fos and c-myc studies provide some biochemical evidence for the importance of protein complex formation in the regulation of ORF destabilizing determinants. A third example o.f a destabilizing sequence in the ORF is the one found in the yeast MATal mRNA (Parker and Jacobson, 1990). These experiments utilized a yeast strain containing a conditional mutation in the yeast RNA polymerase II subunit (Nonet et al., 1987), thereby allowing a direct measurement of mRNA half-lives without the use of chemical transcription inhibitors. Parker and Jacobson (1990) were able to show that the decay of this mRNA required translation through a 65 nt region. This region contains several rare codons, and more recent experiments by Parker and coworkers (Caponigro et al., 1993) have confirmed that it is the presence of rare codons in conjunction with as yet undefined sequences 3' to them that leads to stimulated decay. These authors hypothesize that the pausing or slowing of ribosomal translocation through this region of the mRNA could expose the RNA to the degradative machinery. Whether or not this machinery is associated with the ribosome remains to be determined. What remains unclear from both these and earlier experiments studying the c-fos ORF destabilizers is whether their requirement for translation is equivalent to or superimposed upon the general requirement for translation in mRNA decay. For instance, besides the putative requirement for translation initiation for all mRNA degradation pathways, the requirements of the elements for elongation through them suggests that elongation activates them, possibly by localizing ribosome-associated degradative enzymes to the destabilizing site. That the requirement for mRNA translation could occur at several different points in the decay reaction hints at the potential complexity of the pathway and justifies a more detailed analysis of the relationship between decay and translation. Furthermore, distinguishing between the general and specific requirements for translation in mRNA decay will be essential in discerning the mode of action of different destabilizing elements.
Premature Termination and mRNA Destabilization Investigations on the mechanism by which nonsense mutations within mRNA lead to instability have provided significant insight into the general pathways of mRNA decay. Although it had been appreciated for some time that non-
Cell 416
UPF1
GpppG
GpppG
GpppG
AUG
UAA
AUG
UAA AUG UAA
AUG
(X) UAA AUG UAA
(AAA) n stable
UPF1/, stable
,(AAA)n unstable stable
(AAA) n stable
stable
Figure 3. The Degradationof mRNA ContainingPrematureTermination Codons in Yeast Requiresthe UPF1 Protein and Other mRNA Sequence Elements Deletion of the UPF1 gene (UPFIA) results in the stabihzationof mRNAs containing premature termination codons without affecting the rates of normal mRNA decay. Destabilizationby the termination codonsappearsto requirea downstreamsequencecontaininga methionine codon and the absence of an as yet ill-defined stabilizer sequence(X)just upstream.See Peltzet al. (1993)andthe textfor details.
sense mutations can destabilize mRNA and that their ability to do so decreases as they move further away from the initiator methionine of the message (Losson and Lacroute, 1979), a detailed description of the process had not been achieved. Work on allosuppressors of nonsense mutations in the yeast HIS4 mRNA changed this when it became clear that one of these suppressors, upfl, functioned by stabilizing nonsense-containing messages (Leeds et al., 1991). Studies on other allosuppressors identified in this screen have recently revealed that they also work by stabilizing the mRNA (Leeds et al., 1992). The UPF1 gene product can be deleted from yeast with no alterations in viability on normal medium (Altamura et al., 1992; Leeds et al., 1992). In UPFl-deleted cells, nonsense mRNAs are stabilized back to the levels of their wild-type counterpart (Figure 3). Furthermore, the pathways for general mRNA decay remain unaffected. These data argue that the UPF1 pathway in yeast is distinct from the general mRNA decay pathway and that it is dispensable. However, like the general pathway, nonsensemediated decay requires translation. The stabilization of intron-containing mRNAs in the cytoplasm of upfl mutant cells indicates that besides destroying mutated mRNA, this pathway could be responsible for removing incompletely processed mRNA that may escape retention controls within the nucleus (He et al., 1993). An explanation for why nonsense mutations exhibit a loss of destabilizing activity as they are moved further away from the initiator methionine has recently been presented (Figure 3). By utilizing either wild-type or mutant UPF1 strains, Peltz et al. (1993) were able to show that this distance dependence was created by several distinct RNA sequence elements. First, as expected, the presence of a nonsense mutation was essential for UPFl-mediated decay. Second, the presence of a downstream sequence was also required, and point m utagenesis of this sequence suggested methionine codons were an essential part of it. Finally, the UPF1 pathway was inactivated by the presence of a protection sequence preceding the nonsense mutation. These data are consistent with the model that the transiting ribosome can be made insensitive to nonsense mutations and that sensitivity only occurs when the ribosome
has the ability to reinitiate downstream of the nonsense mutation. Nonsense mutations would therefore lose their destabilizing activity as they move further away from the initiator codon because of the increase in probability of being 3' to a protection sequence and not 5' to another initiator codon. Because the protection sequences are normally found on the RNA, it is probable that they serve another function. One possible role for these sequences would be to prepare the ribosome for termination. If mRNA contains such termination preparation sequences, then it may turn out that the position of ORF destabilizing sequences relative to this sequence may determine how efficiently they control the stability of the message.
3'UTR Destabilizing Sequences Sequences that regulate stability within the 3'UTR of mRNA have received the greatest attention in the mRNA degradation field. The two most well-studied examples are the IRE on the transferrin mRNA and the ARE found on many unstable mRNAs. Taken together, investigations into the mode of action of these two elements have revealed the depth of regulation that probably impinges upon other less well-characterized elements. The regulation of transferrin mRNA degradation in response to changes in cellular stores of iron has been the subject of a recent review (Klausner et al., 1993), so only a brief synopsis of the important findings will be discussed here. Located within the 3'UTR of this mRNA is a region that contains five distinct stem-loop structures capable of binding the IRE-binding protein (IRE-BP). The affinity of this protein for these sequences is what is regulated by cellular iron, and these changes in affinity occur through dissociation and reassociation of an iron-sulfer cluster within the IRE-BP. Interestingly, the IRE-BP also possesses aconitase activity, suggesting an unusual but possibly important linkage between general cellular metabolism and RNA metabolism. The binding of the IRE-BP to the transferrin mRNA stabilizes the mRNA. The mRNA itself is not destabilized owing to the IRE structures, but is instead made unstable owing to the presence of an as yet uncharacterized destabilizing sequence in the vicinity of the stern-loop structures. The current model for how the IRE-BP stabilizes mRNA is that binding to the IREs prevents association of destabilizing factors to the destabilizing sequence. Whether these destabilizing factors are themselves ribonucleases or whether they recruit ribonucleases to destroy the mRNA at a distinct site remains unclear. The regulation of transferrin mRNA decay provides at least two important mechanistic insights. First, it shows how the decay process can be regulated by proteins that can directly respond to changes in the cellular environment. Second, the transferrin mRNA studies reveal that mRNA stabilization may in fact result from an inhibition of an activity recognizing the destabilizing element and that this destabilizing element can be distant from the binding site of the stabilizing proteins. A similar inhibition of mRNA destabilizing sequences by distant sequence elements has been described by Heaton et al. (1992). In this example, a 3'UTR destabilizing
Rewew: mRNA Degradation in Eukaryotes 417
sequence from the yeast STE3 mRNA was found to stimulate degradation of the normally stable yeast PGK1 mRNA only when a portion of the PGK10RF had been removed. The inability of a destabilizing sequence to work in certain contexts raises the possibility that the interaction between different regions of mRNA can lead to increases or decreases in the efficiency of element utilization. Such regulation in cis by DNA sequence elements surrounding transcriptional promoters is well documented, and these data provide some evidence that mRNA metabolism reactions will be subject to similar hierarchical controls. The second well-characterized 3'UTR destabilizing sequence is the ARE. The original observation by Shaw and Kamen (1986) that an ARE in the 3'UTR region of the G M CSF mRNA could stimulate the degradation of the stable 13-giobin mRNA provided much of the basis for current work in identifying destabilizing sequences in general and for understanding the mechanism of AU-rich-stimulated decay in particular. The sequence consensus for the AREs is loosely defined as AUUUA repeated once or several times within the 3'UTR. This pentanucleotide is often found within a U-rich region of the mRNA. Characteristic classes of ARE-containing cellular mRNAs include the lymphokine genes and the immediate early genes. The absence of a clear understanding of the key sequence features of the ARE that allow it to function has precluded a detailed characterization of the involvement of sequences surrounding it. Because of this, the presence of an ARE in a 3'UTR is not sufficient to deem it a destabilizing sequence since it could be inactivated by neighboring sequences. Furthermore, not all AREs are the same. For instance, it has been shown that only a subset of AREcontaining mRNAs are stabilized following changes in cellular metabolism (Lindstein et al., 1989; Schuler and Cole, 1988). A large number of laboratories have examined the proteins bound to the ARE of various mRNAs (Bickel et al., 1992; Bohjanen et al., 1991, 1992; Brewer, 1991; Gillis and Malter, 1991; Hamilton et al., 1993; Savant-Bhonsale and Cleveland, 1992; Vakalopoulou et al., 1991; You et al., 1992). The different methodologies include in vivo or in vitro ultraviolet cross-linking, partial fractionation based upon stimulated decay in in vitro systems, or examination of the differences in sedimentation velocities of RNAs containing AREs and AREs harboring inactivating point mutations. The details of these experiments are too complex to be described here, although several general conclusions can be made. First, it is clear that both nuclear and cytoplasmic proteins can specifically bind to the ARE. The significance of the nuclear-binding proteins remains obscured by the lack of knowledge concerning the contribution of nuclear mRNA processing reactions to the ultimate fate of mRNA in the cytoplasm. Second, the binding activity of some of these proteins appears to increase or decrease in response to the changes in cellular metabolism that lead to alterations in the stabilities of ARE-containing mRNAs. Finally, the existence of a 20S complex on an unstable ARE-containing mRNA and the absence of this complex on ARE-containing mRNAs that are stable owing to ARE inactivation (Savant-Bhonsale and Cleveland,
1992) suggest that ARE activity could be mediated by a complex of proteins (Figure 4). The studies on the ARE-bound proteins are incomplete for several reasons. First, there appear to be two classes of binding proteins associating with the ARE. One class binds equally well to poly(U), while the other appears to be specific for certain AREs. Which class contains proteins that mediate ARE function is not clear. The studies are also incomplete because the relative abundances of each of these proteins remains unknown. For instance, recent data (Hamilton et al., 1993) suggest that many of these proteins may be members of the well-characterized abundant class of heterogeneous nuclear RNA-binding proteins. If true, this would raise serious concerns about the importance of these proteins in destabilization since their cytoplasmic localization has only been detected under conditions of transcriptional arrest (PiSoI-Roma and Dreyfuss, 1992). Because relative abundances are not known, it is unclear whether the cross-linking experiments are detecting specific regulatory proteins or whether general m RNA-binding proteins are showing preferences for either poly(U) or certain AREs. Finally, the studies are incomplete because the absence of an in vitro system in all but one of these studies (Brewer, 1991) results in conclusions based on correlations between changes in cross-linking activity of a protein and measurable changes in mRNA half-lives. Furthermore, no direct confirmation that these proteins mediate ARE function in vivo has been reported. Because of these problems, this group of ARE-binding proteins can only tentatively be considered to be key players in ARE function. Although the role of the ARE-binding proteins remains unclear, the mechanism by which this element stimulates decay has been elucidated in broad outline. Shyu et al. (1991) have convincingly shown that the ARE from the c-fos mRNA mediates decay by first stimulating deadenylation and then providing an element that stimulates the next phase of the degradation process. Uncoupling of the two components of the ARE-mediated response was achieved by point mutations that maintained the ability of
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Figure 4. 20S Complex Formationon mRNAContainingan ARE Occurs Only When the Element Is Active The abmlityof the ARE to stimulate mRNA decay requires that the message be translated, that the ARE not be translated, and that the termination of translationoccurs 5' to the ARE. These requirements can be interpretedto reflect the conditions under which a large 20S particle is formed and maintainedon the mRNA.This particleis potentially the protein complex that mediates the destabilizing properties of the elements. See Savant-Bhonsaleand Cleveland(1992) and the text for details.
Cell 418
the ARE to stimulate deadenylation but inhibited its ability to stimulate mRNA decay. Several other ARE-containing mRNAs have been found that go through a deadenylation and then decay pathway using similar techniques (LairdOffringa et al., 1990; Stoeckle, 1992; Wilson and Treisman, 1988). Together with the work on c-los, these data strongly argue that deadenylation may be a prerequisite for the decay of this class of unstable mRNAs and that deadenylated mRNA is an early intermediate in the degradation process.
Poly(A) Tail Removal and mRNA Degradation How general is the sequential pathway of deadenylation followed by mRNA degradation? Some of the earliest proposals on poly(A) tail function were that the tail provided a buffer to the mRNA from exonucleases and that the pathway of degradation would involve an obligate deadenylation step followed by a continuation of the exonucleolytic degradation in the 3' to 5' direction (reviewed in Peltz et al., 1991). Subsequent experiments investigating the sequential nature of decay reactions for several mRNAs provided ambiguous data. The recent advances in technologies that allow the precise measurement of short poly(A) tails by utilizing RNAase H-directed cleavage of mRNA and the availability of inducible and repressible promoters in several systems (for instance, see Helms and Rottman, 1990; Muhlrad and Parker, 1992; Shyu et al., 1989) have allowed a reexamination of whether mRNA must be deadenylated before it is degraded. A systematic study of the timing of deadenylation relative to the decay of several yeast mRNAs utilizing the yeast activatable and repressible galactose promoter has revealed that the mRNAs are deadenylated before they are degraded (Decker and Parker, 1993). These data are consistent with the effects of two conditional mutations in yeast that lead to an enhancement of both poly(A) and mRNA destabilization (Minvielle-Sebastia et al., 1991). They are also consistent with the data of Wellington et al. (1993) showing that ORF destabilizers induce deadenylation. The experiments by Decker and Parker (1993) are surprising since the mRNAs examined exhibit the full range of stability in the cell. As a result, they suggest that the pathway of deadenylation preceding decay may not be limited to just the highly unstable messages. These data are an extension of work studying the degradation pathway of the yeast mating factor ~2 (MFA2) mRNA (Muhlrad and Parker, 1992). In this work, a destabilizing sequence within the 3'UTR of the message was found to behave in a fashion analogous to the ARE from higher cells in that it stimulated the degradation of the mRNA by stimulating its deadenylation. Importantly, mutations that inhibited the rates of MFA2 deadenylation also decreased the rates of mRNA decay. This work highlighted the importance of distinguishing between the shortening of poly(A) tails down to approximately 12-15 residues and the shortening of these oligoadenylate tails to lengths below 10 nt in a process called terminal deadenylation (see below). The recent study by Decker and Parker (1993) also iden-
titles an in vivo cleaved mRNA degradation intermediate that shows temporally correct precursor-product relationships. Previous studies had only detected intermediates in a steady-state population of mRNA (Binder et al., 1989; Lim and Maquat, 1992; Stoeckle, 1992; Stoeckle and Hanafusa, 1989; Vreken and Rau~, 1992). This degradation intermediate was stabilized by a 5' tract of G residues, which had been shown previously to protect mRNA fragments from 5' exonucleases (Vreken and Rau~, 1992). The intermediate was a fragment of mRNA containing the 3' end of the molecule with several adenines at the tail. The existence of this intermediate in vivo shows that although deadenylation is a prerequisite for initiating mRNA decay, the subsequent nucleolytic reactions can occur within the message.
Pathways of mRNA Degradation and Trans-Acting Factors Using the data summarized in this review, a general model for mRNA degradation can be created (Figure 5). mRNAs are not degraded until their tails are shortened to lengths below 10 nt, a length that is incapable of high affinity binding to the poly(A)-binding protein (see below). The regulation of mRNA decay can result from alterations in the rates of poly(A) tail shortening or terminal deadenylation since poly(A) removal appears to be rate limiting in the decay process. Once deadenylation is completed, there is a rapid degradation of the mRNA that occurs by either endo- or exonucleases. The coupling of the activation of these nucleases, which remain undefined, to the completion of deadenylation explains why it is so difficult to find intact but terminally deadenylated mRNA within the cell.
GpppG
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-
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Figure 5. DeadenylationMay Be a Prerequisitefor mRNA Degradation Processiveshorteningof poly(A)tails by the normallydistributivepoly(A) nucleasecould be inducedby mRNAdestabilizingsequences. The rate of terminaldeadenylatloncouldalso be regulatedby these elements. Oncedeadenylated,the mRNAcan be destroyedby many differenttypesof ribonucleases.Theactivationof the degradationstep is tightlycoupledto the completionof deadenylation,and as a result alterations in deadenylationrates will changemRNA half-lives.See the text for details.
Review:mRNADegradationin Eukaryotes 419
The degradation pathway of mRNAs containing a premature termination codon is probably different than that for normal mRNAs. One indication of this is the lack of requirement for the UPF1 pathway for yeast cell viability and the lack of effect of the UPF1 deletion on normal mRNA decay rates. Furthermore, Shyu et al. (1991) have found that I~-globin mRNA containing a destabilizing nonsense mutation is degraded before it is deadenylated. The mechanism of action of destabilizing sequences within mRNA can be rationalized from these data. These sequences require translation for activitysince their recognition is mediated by factors brought to the mRNA by the translational machinery. Once recognized, the elements induce decay by activating poly(A) tail removal. Following the terminal deadenylation step, identical or other sequence elements within mRNA are recognized by the exoor endonucleases that eventually lead to the destruction of the mRNA. Several important predictions can be made from this model. First, if deadenylation is a common initial step in the degradation pathway of mRNAs, then the target of each of the destabilizing sequences may be to activate the single event of deadenylation. As a result, an understanding of destabilizing sequence activity will come through examining how they activate this process. Second, this model predicts that the identification of decay intermediates in vivo or in in vitro reactions will not necessarily give insight into what the rate-limiting step in the decay process is. For instance, endonucleolytic cleavage of deadenylated mRNA may be much faster than deadenylation, and as a result the intact deadenylated intermediate will not be seen owing to its rapid metabolism. Finally, this model predicts that elements within an mRNA can effect degradation events occurring elsewhere on the mRNA. If deadenylation is a key step in the regulation of mRNA stability, then an understanding of the biochemistry of this step will be essential for a dissection of decay pathways. mRNA polyadenylation occurs in the nucleus of cells, and the posttranscriptionally synthesized tail is typically homogeneous in length, ranging from 70-90 nt in yeast to 220250 nt in mammalian cells (reviewed in Sachs, 1990). Following transport of the mRNA, the long poly(A) tail is shortened in the cytoplasm. In yeast, this shortening reaction requires the presence of a specific protein bound to the poly(A) tail, the poly(A)-binding protein, for full efficiency (Sachs and Davis, 1989), and it is this reaction that could be regulated by the different destabilizing sequences on many RNAs. As described above, the poly(A) shortening reaction can be separated into two phases, the first being the shortening of the tail down to 12-25 residues and the second terminal deadenylation step being the removal of some or all of these residues. The ribonuclease involved in poly(A)-binding proteindependent shortening of poly(A) tails from yeast has been purified and cloned (Sachs and Deardorft, 1992). This poly(A) ribonuclease (PAN) is unique among identified eukaryotic RNAases in that it requires a protein-RNA complex as a substrate. Conditional mutations in PAN reveal
it is also a translation initiation factor. PAN deadenylation activity in vitro exhibits the two phases of shortening seen in vivo, and both the shortening phase and the terminal deadenylation phase can be regulated by 3'UTR sequences in a reconstituted system containing highly purified enzyme, poly(A)-binding protein, and synthetic RNA (Lowell et al., 1992). In this system, the stimulation of PAN shortening activity by sequence elements within the 3'UTR of the yeast MFA2 mRNA occurs by switching PAN from a distributive to a highly processive enzyme. The mechanism by which this switch occurs is under investigation, but it is probably analogous to the mechanism by which AREs (and potentially other destabilizing sequences within the mRNA) stimulate deadenylation of the poly(A) tail. The inhibition of the terminal deadenylation step by a sequence element within the 3'UTR of a synthetic mRNA in the in vitro system suggests that poly(A) tail stabilization by sequence elements within stable messages could also be occurring in vivo. The characterization of PAN reveals some interesting properties that may become more general for RNA degradation reactions. First, the substrate for the ribonuclease is not naked mRNA, suggesting that the search for RNAases that specifically recognize sequences within mRNA that have been identified as destabilizing sequences may be unsuccessful unless the proper ribonucleoprotein substrate is identified. That the nucleolytic substrate could be a ribonucleoprotein complex negates the simple hypothesis that RNA-binding proteins will always protect mRNA from decay. Second, PAN activity can be regulated from distant sites. This argues that destabilizing sequences may not harbor nucleolytic sites, but instead target sites elsewhere on the mRNA for degradation. A corollary of this is that the identification of endonucleolytic cleavage sites on RNA decay intermediates may not yield information about what region of the mRNA made these sites nuclease sensitive. Furthermore, the ability of PAN to be activated or inhibited over distances in vitro is consistent with the hypothesis that destabilizing sequences located throughout an mRNA molecule can affect the deadenylation and decay pathways. Last, the requirement for PAN in both translation and (presumably) RNA decay in vivo leads to the suggestion that some of the factors involved in mRNA decay may also be complexed with translation initiation factors. This conclusion is consistent with the apparent requirement for translation in mRNA decay discussed above. Other in vitro systems have also been utilized to explore mRNA decay pathways, but none have utilized pure components. The most successful in vitro reactions appear to be those that examine the degradation of mRNA attached to polyribosomes (Brewer, 1991; Vreken et al., 1992). Interestingly, the mRNAs in these experiments appear to be degraded, even though the polyribosomes are incubated under conditions that do not allow translation. The significance of this may only become clear after further work examining the in vivo requirements for translation. Finally, a mammalian poly(A)-specific nuclease has been partially purified (,~,strom et al., 1992), although the relationship of
Cell 420
this enzyme to the yeast PAN and its importance in mRNA decay reactions are not yet known.
Summary and Perspectives mRNA contains within it the information needed to determine its stability within the cell. Destabilizing information lies within discrete regions of sequence, and these sequence elements can be found throughout the message and may be redundant. Many destabilizing elements require translation for activity, although the reasons for this are not understood. The masking or activation of these elements by other proteins probably leads to regulated mRNA decay rates. The target of these elements could be the deadenylation machinery since many mRNAs are only degraded after the rate-limiting step of deadenylation is completed. Once the mRNA is deadenylated, these or other elements may provide nucleolytic sites that allow for the rapid degradation of the mRNA. A more complete understanding of mRNA decay will in part require more detailed descriptions of the sequences within m RNAs that determine instability. Equally important will be a thorough in vivo analysis of how an mRNA is degraded, an understanding of why there is a requirement for translation, and the creation of in vitro RNA decay systems that accurately reflect the in vivo decay pathways. Current work in the field utilizing a combination of biochemical and genetic approaches will give insight into each of these areas, and with this should come the discovery and subsequent elucidation of the regulatory networks impinging on these processes.
Acknowledgments I thank Allan Jacobson and Matthew Sachs for critical reading of the review and each of the laboratories that provided preprints of their manuscripts. Work in the laboratory of the author is supported by National Institutes of Health grant R29 GM-43164 and in part by awards from the Markey, Searle Scholars, and March of Dimes Foundations.
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