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Tales of poly(A): a review
Oene. 91 (1990) 151-158 Elsevier
151
GENE 03585
Tales o f poly(A): a review (Protein synthesis; poly(A)-binding protein; 3'-translational enhancer)
David Munroe* and Allan Jacobsen Department of Molecular Genetics and Microbiology. University of Massachusetts Medical School. Worcester. MA 01655 (U.S.A.) Received by M.P. Wickens: 8 February 1990 Accepted: 16 March 1990
SUMMARY
Until recently, evidence to support a translational role for the Y-poly(A) tract of eukaryotic mRNAs has been mostly indirect, including: a correlation between the adenylation status of individual mRNAs and their translatability in vivo or in vitro, the demonstration that exogenously added poly(A) is a potent competitive inhibitor of the translation of poly(A) + mRNA, but not poly(A)- mRNAs in vitro, and a correlation between the abundance and stability of poly(A)-binding proteins (PABPs) and the rate of translational initiation in vivo. However, more recent studies demonstrate directly that poly(A) + mRNAs can initiate translation more efficiently than poly(A)- mRNAs, and indicate that this effect is: (i) targeted to the formation of 80S initiation complexes, and (ii) likely to be mediated by the cytoplasmic PABP. We suggest that the 3'-poly(A) tail should be considered a translational enhancer which may stimulate translational initiation in much the same way that transcriptional enhancers are thought to stimulate transcriptional initiation.
INTRODUCTION
Several recent reports, documenting abrupt changes in the poly(A) tail length of individual mRNAs or mRNA populations in parallel with changes in their translatability during development (Carrazana et al., 1988; Hyman and Wormington, 1988; Paynton et al., 1988; Robinson et al., 1988; Strickland et al., 1988; Zingg et al., 1988; Carter and Murphy, 1989; McGrew ctal., 1989; Vassalli et al., 1989), Correspondenceto: Dr. A. Jacobson, Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 (U.S.A.) Te!.(508)856-2442; Fax (508) 856-5920. * Current address: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.) Tel. (617)253 3020. Abbreviations: aa, amino acid(s); bp, base pair(s); GI0, uncharacterized
have generated renewed interest in the function of poly(A) and its possible role in gene regulation. Since the discovery of mRNA poly(A) tails almost two decades ago (Kates, 1970; Lim and Caneilakis, 1970; Darnell et al., 1971a,b; Edmonds et el., 1971; Lee et al., 1971) three general models for their function have been proposed. These postulate a role for poly(A) in mRNA processing and transport, mRNA stability, or protein synthesis. In this review we will focus on the evidence which implicates a role for poly(A) in protein synthesis, and in particular, a role in the regulation of mRNA translational efficiency. (See recent reviews by Jacob et al., 1990, and Bernstein and Ross, 1990, for discussions ofpolyadenylation and of tthe possible role of poly(A) in mRNA stability). GENERAL DISCUSSION
Xenopusmaternal mRNA; kb, kilobase(s) or 1000 bp; mRNP, messenger ribonucleoprotein particle; nt, nucleotide(s); PABP, poly(A)-bindingprotein; poly(A) ÷ RNA, RNA containing a Y-poly(A) tract; poly(A)- RNA, RNA lacking a Y-poly(A) tract; r, ribosomal; RNP, ribcnueleoprotein particle; spb, yeast gene encoding suppressors of PABP mutations; tPA, tissue-type plasminogen activator; UTR, untranslated region. 0378-1119/90/$03.50 © 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)
(a) Correlations between the adenylation status of mRNA and its translatability in vivo and in vitro In addition to the studies alluded to above, there are numerous other experimental systems (especially develop-
152 ing systems) in which a direct correlation has been observed between the polyadenylation status ofmRNA and its translational efficiency (i.e., extent of polysome loading) in vivo. Perhaps the most well known of these correlations involves the fate of five developmentally regulated mRNAs upon fertilization of Spisula oocytes (Rosenthal et a1., 1983). Four of these transcripts are poly(A)-mRNAs and are translationally inactive in the oocyte. Following fertilization, these mRNAs becorae adenylated and are simultaneously recruited onto polysomes. In contrast, a fifth transcript, a-tubulin mRNA, is deadenylated at fertilization, coincident with its exclusion from polysomes, Similar examples of the efficient translation of poly(A)+mRNAs and the inefficient translation of poly(A)-mRNAs, and of the interconversion between these states as a consequence of developmental or environmental stimuli, have been observed in species as diverse as Dictyostelium dbcoideum and rats (summarized in Table I). The relationship between poly(A) tail length and translational efficiency in vivo is not absolute. There are examples of translationally inactive polyadenylated mRNAs (Raft, 1980; Rosenthal etal., 1983) as well as examples of mRNAs which appear to lose their poly(A) tracts as they become translationally active (Hruby and Roberts, 1977; latrou and Dixon, 1977; Kleene, 1989). The possibility that 3'-poly(A) tails might play a role in translation has been extensively tested in several in vitro systems. Ira early studies, a relationship between 3'-poty(A) tails and translation was tested and 'ruled out' by experiments which compared the in vitro translational capacity of adenylated and deadenylated forms of various mRNAs. It was generally concluded that artificial deadenylation (Bard
eta!., 1974; Sippel etal., 1974; Soreq etal., !974; Williamson et al., 1974; Spector et al., 1975) or klockage of the poly(A) tail with poly(U) (Munoz and Darnell, 1974) did not significantly reduce the translatability of mRNA. Support for a translational role for poly(A) was subsequevfly resurrected by Doei and Carey (1976) who noted h'mt the translation systems used in the previous studies reinitiated poorly and were thus relatively insensitive to differences in mRNA translational efficiencies. Doel and Carey (1976) were able to show that, while native ovalbumin poly(A)+mRNA was translated more efficiently than its deadenylated counterpart in a highly active reticulocyt,~ extract, no such difference was seen in less active wheat-germ extracts. This translational discrimination was attributed to a reduced rate of initiation on the poly( A ) - m R N A since this mRNA was found on smaller polysomes and participated in fewer rounds of translation. The translational efficiency of mRNAs microinjected into Xenopus oocytes or transferred to plant protoplasts by electroporation also correlates with adenylation status. For example, Deshpande etal. (1979) reported that native ohfglobulin mRNA [average poly(A)~Ts] was translated more efficiently and reached peak translatability faster than poly(A)-poor a2fglobulin mRNA [average poly(A)4o] in microinjected Xenopus oocytes. Drummond et al. (1985) found that synthetic poly(A) + mRNAs, encoding chicken lysozyme, calf preprochymosin, and Xenopus/~-globin were translated 5-20-fold more effectively than poIy(A)-mRNA. Galili et al. (1988) have demonstrated a similar translational discrimination following microinjection of synthetic maize zein or Xenopus/~-globin transcripts into Xenopus oocytes. Furthermore, they reported that the
TABLE 1 Correlations of poly(A)tail length and translationalefficiencyin vivo Organism/conditions
mRNA
Correlation"
Reference
Dlctyostellumdiscoideum/development Urechbcaupo/oogensis
Vegetative and developingmRNAs Maternal mRNAs Total mRNA hsp90 mRNA Different mRNAs
A and B A and B A and B A A and B
Pa]atnik eta]. (1984); Shapiro eta]. (1988) Rosentha] and Wilt (1986) Nemer eta]. (1975) Bedard and Brandhorst (1986) Rosentha] et al. (1983); Rosenthal and Ruderman (1987) Hyman and Wormington (1988) McGrew eta]. (1989) Restifo and Guild (1986)
Sea urchin embryo Sea urchin/oogenesis
Spbula/fertilization
Xenopuslaevbloogenesis r-protein mRNAs Xenopuslaevb/oogenesis GI0 mRNA Drosophila melanogaster/prepupalsalivary Different mRNAs gland develcpment Mouse/oogenesis
B A B
Mouse/oogenesis
Different mRNAs Tissue plasminogenactivator mRNA
A and B A
Rat/circadian rhythm Rat/osmotic stress
Vasopressin mRNA Vasopressin mRNA
A and B A and B
Paynton eta]. (1988) Huarte eta]. (1987); Strickland eta]. (1988); Vassa]li eta]. (1989) Robinson eta]. (1988) Carrazana eta], (1988); Zingg eta]. (1988); Carter and Murphy (1989)
" A, adenylationof mRNA correlates with recruitmentonto polysomes;B, deadenylationof mRNA correlates with exclusionfrom polysomes.
153 poly(A) + mRNAs were found on polysomes with a larger average number of ribosomes than their unadenylated counterparts, indicating that the poly(A)-mRNAs were. deficient in translational initiation. In off,er experiments; synthetic poly(A) + RNA, encoding r-protein L 1, was found to be actively translated following microinjection into stage VI oocytes. Upon oocyte maturation these mRNAs became deadenylated and released from polysomes, paralleling the deadenylation and translational inactivation of endogenous r-protein mRNAs (Hyman and Wormington, 1988). Recently, Gallic etal. (1989) have investigated the translational role of Y-POly(A) tracts in plants. They observed that a synthetic u-glucuronidase (A)~omRNA is translated 16-120-fold more effectively than a similar poly(A)-mRNA in electroporated tobacco, carrot, maize, and rice protoplasts. (b) The cytoplasmic poly(A)-hinding protein (PABP): indirect evidence that it has a role ia translation and that it is a mediator of poly(A) function The mRNAs of every eukaryotic cell are found in ribonucleoprotein complexes (mRNPs), intimately associated with as many as 20 different polypeptide species (Irwin et al., 1975; Jeffery, 1977; Mirkes, 1977; Jain and Sarkar, 1979; Greenberg, 1980; Adams et al., 1981; Setyono and Greenberg, 1981; Moon, 1983; Manrow and Jacobson, 1986; 1987; Bandziulis et al., 1989). By far, the most widely studied of the mRNP proteins are those which associate with the poly(A) tract of mRNA, the PABPs. First described by Blobel (1973), PABPs have been found in every eukaryotic cell examined thus far. Sequence analysis has revealed extensive evolutionary conservation of the yeast, XenoDus, and human PABP-encoding genes (Adam et al., 1986, Sachs et al., 1986; Grange et al., 1987; Zelus et al., 1989). Biochemical analysis of purified yeast PABP has established that: (1) the packing density is approx. 1 PABP per (A)2s (in striking agreement with the size of poly(A) protected from nuclease digestion by PABP; Baer and Kornberg, 1980; 1983); (2) the minimum length of poly(A) required for efficient binding to PABP is 12nt; and (3) PABP can bind to (A)!80_220, and the A-rich domain of the 5'-untranslated region of the PABP mRNA with equal efficiency (Sachs et al., 1986; 1987). Deletion analyses have shown that the PABP is essential for cell viability (Sachs et al., 1986; 1987). Both the evolutionary conservation of the PABP and its necessity for cell viability obviously reflect an important cellular function. Earlier experimental evidence suggested that PABP may mediate the translational effect of 3'-poly(A) tracts. The first such data was provided by Jacobson and Favreau (1983) who reasoned that if 3'-poly(A) tracts did function in protein synthesis then exogenous poly(A) mig,ht competitively inhibit the in vitro translation of poly(A) + mRNAs in
much the same way that 5' -cap structures inhibit the in vitro translation ofcapped mRNAs (Hickey et al., 1976; Roman et al., 1976). They found that exogenous poly(A) inhibited the translation of poly(A)+mRNA, but not poly(A)-mRNA, in reticulocyte lysates and that comparable inhibition was not observed with other polyribonucleotides (Jacobso~l and Favreau, 1983). This poly(A)-mediated inhibition was dependent upon the size of the competitor poly(A) and could be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. Furthermore, poly(A)-mediated inhibition did not alTectthe average size of the polypeptides synthesized, suggesting that this inhibition occurs at the level of translational initiation. These observations have been confirmed by others in reticulocyte lysates (Bablanian and Banerjee, 1986; Grossi de Sa et al., 1988), L-cell lysates (LeMay and Millward, 1986), dry pea-seed extracts (Sieliwanowicz, 1987) and in Xenopusoocytes (Drummond et al., 1985). The most straightforward and generally accepted interpretation of these results is that exogenous poly(A) inhibits translation by fimiting the availability of unbound PABP. Consistent with this interpretation, the addition of purified PABP has been reported to overcome poly(A)-mediated translational inhibition in reticulocyte iysates (Grossi de Sa et al., 1988). These experiments do not, however, exclude the possibility that exogenous poly(A) has a high affinity for another component of the translation apparatus and that addition of purified PABP simply competes for this interaction. Other data supporting a role for the PABP in translation have been provided by Manrow and Jacobson (1986: 1987). They found that, during the first 30 min of DictyosteIium discoideum developm~,~t, when translatic~nal initiation is sharply reduced (Cardelli et al., 1981), the PABPs are selectively degraded (Manrow and Jacobson, 1986). The turnover of the PABPs increased 20-25-fold at this time, resulting in an 80-.90% decrease in their relative abundance. In related experiments (Manrow and Jacobson, 1987), similar reductions in PABP stability and abundance were found to accompany a heat-shock induced reduction in translational initiation. In contrast, the level of PABPs was not affecte~ by reductions in the rate of translational elongation (Manrow and Jacobson, 1987). Additional indirect evidence for a translational role for the PABP has been obtained by Sieliwanowicz (1987) who showed that the translational activity of an in vitro translation system derived from the embryo axes of dry pea seeds is directly related to the relative abundance of the pea 60-kDa PABP. (c) A model for poly(A) function and recent evidence to support it The data discussed thus far, although indirect, are consistent with a translational model for poly(A) function
154
(Jacobsen and Favreau et al., 1983; Palamik ot al., 1984) which postulates that: (l)an interaction between the 3'-poly(A)tract of mRNA and a cytoplasmic PABP specifically enhances translational initiation; (2)this enhancement is directly dependent upon the length of the poly(A) tail such that mRNA with relatively long poly(A) tails has a translational advantage over mRNA with shorter poly(A) tails; (3)this enhancement is not essential for translation; and (4)the regulatory mechanisms which ensure the efficient translation ofpoly(A) + mRNA vs. poly(A)- mRNA may be quite different. Four recent reports (Sachs and Davis, 1989; 1990; Vassalli et al., 1989; Munroe and Jacobsen, 1990) give credence to this model and provide direct evidence for the role played by both poly(A) a~ld PABP in protein synthesis. Those data confu'm thst poly(A) + mRNAs have a translational advantage over poly(A)-mRNAs and establish that: (1) the effect of poly(A) on translation is directly dependent upon its length; (2)the effect of poly(A) on translation is mediated by the PABP; and (3) the poly(A)/PABP complex is involved in translational initiation and, more specifically, in the joining of 60S ribosomal subunits to 48S preinitiation complexes during the formation of 80S initiation complexes. Sachs and Davis (1989) have utilized a genetic approach to demonstrate that the PABP is involved in translational initiation. These experiments involved in vivo depletion of PABP in Saccharomyces cerevisiae by promoter inactivation or by the use of a PABP temperature-sensitive mutation. Depletion of PABP resulted in a marked decrease in the amount of polysomes along with a concomitant increase in free r-subunits, a phenotype characteristic of a translational initiation defect. Seven indepezident, extragenic revertants of the ts mutation which allow translational initiation in the absence of PABP wore isolated. All of these revertants affected cellular levels of the 60S r-subunit. Two revertants (s,vb2-1 and spb4-1) were characterized further and localized, respectively, to the gone for the 60S r-protein L46 and to a gone encoding a putative rRNA helicase involved in the maturation of 25S rRNA (Sachs and Davis, 1989; 1990). Those data indicate that translation in the absence of functional PABP requires an alteration in the structure of the 60S r-subunit. Vassalli et al. (1989) have examined the correlation between polyadonylation and translational ac~vation of ~PA mRNA during meiotic maturation in the mouse oocyte. Microinjection experiments utilizing various tPA chimeric and 3'-blocked in vitro synthesized mRNAs have demonstrated that: (1) this regulated polyadenylation requires sequences present in the 3'-untranslated region of tPA mRNA, and (2) the presence of a long poly(A) tract is both necessary and sufficient for the observed translational recruitment oftPA mRNA. These data show that the trans-
lational discrimination of tPA mRNA before and after germinal vesicle breakdown is due solely to changes in the polyadenylation status of mRNA. Munroe and Jacobsen (1990) have directly compared the translatability of synthetic mRNAs, differing solely in 5'-cap and/or 3'-poly(A) structures, in a cell-free rabbit reticulocyte translation extract. The results of such experiments confirm that poly(A)- mRNAs have a reduced translational capacity and that the effect of poly(A) on translation is directly related to its length. A direct comparison of the polysomes formed by poly(A)+-mRNAs and poly(A)-mRNAs revealed that poly(A)-mRNAs are recruited onto polysomes to a lesser extent than poly(A) + mRNAs and that the polysomes which are formed by poly(A)-mRNAs contain fewer ribosomes than those formed by poly(A) + mRNAs. These data, together with the finding that translational elongation rates along adenylated and unadenylated mRNAs are identical, establish that mRNA poly(A) tails function in translational initiation. Other experiments showed that the defect in poly(A)-mRNAs is distinct from that associated with capdeficient mRNAs and results in a reduced ability to.join 60S r-subunits to 48S preinitiation complexes during the later stage(s) of translational initiation. Utilizing a label transfer protocol, Munro¢ (1989) also showed that the major difference in the mRNP proteins associated with poly(A)+mRNAs or poly(A)-mRNAs is due to proteins which associate with poly(A). Although proteins larger than PABP monomers were detected in these experiments, their Mrs are suggestive of PABP dimers and trimers. The appearance of such putative dimers and trimers increased as a function of poly(A) tail length in a manner consistent with expectatioos based on binding site sizes and in parallel with increases in translational efficiency. These results complement the genetic studies of' Sachs and Davis (1989) and suggest that the functional form of poly(A) is the poly(A)/PABP complex. In other experiments, mRNAs with or without caps and/or poly(A) tails were shown to be differentially sensitive to translational inhibition by exogenous competitor poly(A) (Munroe and Jacobsen, 19901. As expected, the translation of` poly(A) ÷ mRNAs was markedly reduced by the presence of` exogenous poly(A). In contrast, the translation of`capped, unadenylated mRNAs was actually stimulated by exogenous poly(A). No such stimulation was seen with uncapped, unadenylated mRNA implying that: (1) poly(A) may be capable of working in trans to augment the translation of capped, unadenylated mRNAs and (2)event(s) in translational initiation involve the recognition of both 5'.cap and 3'-poly(A) structures (together with their associated proteins). [These observations may also provide a clue to the function of the abundant oligo(A) molecules present amongst the genomic RNAs of
155 reoviruses (Carter et aL, 1974; Stoltzfus et al., 1974). Oligo(A) associated with reovirus particles may act to suppress the translation of capped, host-cell poly(A) ÷ mRNAs while at the same time stimulate the translation of reoviral mRNAs which are normally capped, but poly(A)- ]. Collectively, the experiments of Sachs and Davis (1989; 1990), Munroe (1989), and Munroe and Jacobsen (1990) demonstrate that the poly(A)/PABP complex, and not poly(A) or PABP alone, has a role in the formation of 80S initiation complexes. Such results are consistent with all of the correlative evidence discussed above and also with the observation by Nelson and Winkler (1987) that, in reticulocyte lysates, histone poly(A)- mRNA forms a significantly higher percentage of 'half-mers' (mRNAs bound to 40S r-subunits but lacking 60S r-subunits) than globin poly(A) + mRNA. (d) How is poly(A)/PABP involved in the regulation of gene expression.* Having discussed evidence supporting the involvement of poly(A)/PABP in translational initiation we would now like to speculate about how this complex might function in the regulation of gene expression. Such regulation can be grouped into two broad categories: (l) discrimination between 'old' and 'new' mRNAs, and (2)targeted translational regulation via the manipulation of mRNA adenylation status and/or PABP abundance or structure. mRNA poly(A) tails are heterogeneous in length, ranging, in mammals, from a maximum of (A)25o on newly synthesized mRNA (Brawerman, 1976), to a minimum of about (A)3o (Sheiness and Darnell, 1973; Ahlquist and Kaesberg, 1979). Siace the activity of poly(A) in translation is directly related to its length (Jacobsen and Favreau, 1983; Munroe and Jacobsen, 1990), it would seem logical to predict that newly synthesized mRNA, with relatively long poly(A) tracts, should have a translational advantage over preexisting mRNAs with relatively short poly(A) tracts. Preferential translation of the most recently transcribed sequences would ensure the rapid synthesis of their encoded products. Evidence for translational discrimination between 'new' and 'old' mRNAs has been obtained in Dictyostelium (Palatnik et al., 1984; Shapiro et al., 1988). Likewise, a change in the adenylation status of individual mRNAs or mRNA populations may be sufficient to induce rapid changes in protein synthetic patterns in the absence of significant mRNA synthesis or turnover (Palatnik et al., 1984). As discussed earlier, there are several examples of dramatic changes in translational capacity accompanied by concomitant changes in mRNA adenylation status (Table I). The majority of these changes occur in developing systems, where rapid changes in the pattern of protein synthesis are often necessary. Since such adenylation/ deadenylation may involve only a subset of the cellular
mRNAs (e.g., Sp/sula fertilization; Rosenthal et al., 1983; Rosenthal and Ruderman, 1987), the signals which target individual mRNAs for adenylation and/or deadenylation must be tightly regulated and highly specific. Well-characterized examples of these signals have recently been reported by McGrew et al. (1989) and Vassalli et al. (1989) who identified short, c/s-acting 3'-UTR sequence elements that apparently direct the polyadenylation and subsequent polysomal recruitment of the G 10 and tPA mRNAs during oocyte maturation in Xenopus/aev/s and mice, respectively. The translational effect of poly(A) is almost certainly mediated by the PABP (Sachs and Davis, 1989; 1990; Munroe, 1989). Not surprisingly, changes in the stability and abundance of this protein coincide with changes in the rate of translational initiation which occur during development (Manrow and Jacobsen, 1986) or heat shock (Manrow and Jacobsen, 1987). Similarly, structural variants of the PABP which have retained poly(A)-binding activity, but lost stimulatory activity, may be expected to act as translational repressors. A candidate for such a variant is an oocyte-specific PABP (Swiderski and Richter, 1988) which appears to act as a translational inhibitor (Kick et al., 1987). Thus, the hyper-polyadenylation of'stored' mRNAs following fertilization in Xenopus (McGrew et al., 1989) may occur to provide an accessible binding site for the 'stimulatory form' of PABF. (e) Current models for italy(A) function Verification of the involvement of the poly(A)/PABP complex in translational initiation has led to the proposal of two new models regarding poly(A) function. Sachs and Davis (1989) have proposed that co-recognition of 5'- and 3'-mRNA structures (i.e., caps, poly(A) tails, and their associated proteins) during the initiation of translation may occur simply to prevent the attempted translation of partially degraded mRNAs. On the other hand, Munroe and Jacobson (1990) have proposed that poly(A) sequences be considered translational enhancer sequences which stimulate translational initiation in much the same way that transcriptional enhancers are thought to stimulate the initiation oftranscription (Ptashne, 1986; 1988).These models are not mutually exclusive and both predict that the mRNA 3' and 5' termini interact during translational initiation (Fig. 1). Evidence supporting such an arrangement includes mRNA secondary structure maps which place the mRNA 5' and 3' domains in close proximity (Heindell et al., 1978; Lockard et al., 1986), electron micrographs of polysomes with interacting 5' and 3' ends (Warner etal., 1962; Dubochet etal., 1973; Hsu and Coca-Prados, 1979; Ladhoff eta]., 1981; Christensen etal., 1987), and the demonstration that exogenous poly(A) may be capable of working in trans to stimulate the translation of capped, poly(A)-mRNA (Munroe and Jacobsen, 1990). Con-
156
cap
j
(1) poly(A) is an enhancer ofthe formation of 80S translational initiation complexes; (2) the effect of poly(A) on translation is directly related to its length; (3) the mediator of the function of poly(A) is the cytoplasmic PABP.
ACKNOWLEDGEMENTS Fig. 1. A model for the role of Y-poly(A) tracts in the initiation of translation. We propose that poly(A) acts as the formal equivalent of a transcriptional enhancer. The cartoon depicts the 3'.poly(A): PABP complex promoting the formation of 80S initiation complexes at the mRNA 5'-end (either by a direct interaction with the 60S r-subunit or by facilitating the binding of other protein(s) which, in turn, influence the binding ofthe 60S r-subunit), cap, methylated, inverted nucleotide at the 5' terminus of the mRNA; AAAAA: poly(A) tract at the 3' terminus of the mRNA.
sistent with the enhancer model, is the t'mding that, at low levels, exogenous PABP acts to stimulate in vitro translation of poly(A) + mRNAs while larger amounts exhibit an inhibitory or 'squelching' effect (Sieliwanowicz, 1987). Transcriptional enhancers are known to be orientationindependent. Similarly, the enhancer model for poly(A) function would predict that the location of poly(A) sequences on the mRNA would be unimportant. In this regard, there are several known mRNAs which contain 5'-poly(A) sequences which should be capable of binding PABP, including poxvirus mRNAs (Bertholet et al., 1987; Patel and Pickup, 1987; Schwer et al., 1987) and PABP mRNAs (Adam et al., 1986; Sachs et al., 1986; Grange et al., 1987). The apparent conservation of the 5' A-rich domain between yeast and human PABP mRNAs and amongst the mRNAs of the members of the poxvirus family suggests that it provides some advantage. One possible advantage would be the enhancement of translational initiation even on relatively 'old' mRNAs with shorter 3'-poly(A) tracts. Consideration of the poly(A) tail as a translational enhancer raises the possibility that other sequences in the 3'-UTR also have a translational regulatory role. We anticipate that crosslinkingbstudies and further characterization of the spb mutants ('Sachs and Davis, 1989; 1990) will identify polypeptides which are the targets of PABP function or which stimulate or repress PABP activity. Amongst such polypeptides may be mRNA-binding proteins which, like the PABP, usually bind to sites within the 3'-UTR. (g) Conclusions Recent experiments demonstrate that the 3'-poly(A) tract of eukaryotic mRNAs has a role in translational initiation. These experiments lead us to conclude that:
Work in the authors' laboratory is supported by a grant (GM27757) from the National Institutes of Health. We thank B. Lewin for not letting us down.
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151
GENE 03585
Tales o f poly(A): a review (Protein synthesis; poly(A)-binding protein; 3'-translational enhancer)
David Munroe* and Allan Jacobsen Department of Molecular Genetics and Microbiology. University of Massachusetts Medical School. Worcester. MA 01655 (U.S.A.) Received by M.P. Wickens: 8 February 1990 Accepted: 16 March 1990
SUMMARY
Until recently, evidence to support a translational role for the Y-poly(A) tract of eukaryotic mRNAs has been mostly indirect, including: a correlation between the adenylation status of individual mRNAs and their translatability in vivo or in vitro, the demonstration that exogenously added poly(A) is a potent competitive inhibitor of the translation of poly(A) + mRNA, but not poly(A)- mRNAs in vitro, and a correlation between the abundance and stability of poly(A)-binding proteins (PABPs) and the rate of translational initiation in vivo. However, more recent studies demonstrate directly that poly(A) + mRNAs can initiate translation more efficiently than poly(A)- mRNAs, and indicate that this effect is: (i) targeted to the formation of 80S initiation complexes, and (ii) likely to be mediated by the cytoplasmic PABP. We suggest that the 3'-poly(A) tail should be considered a translational enhancer which may stimulate translational initiation in much the same way that transcriptional enhancers are thought to stimulate transcriptional initiation.
INTRODUCTION
Several recent reports, documenting abrupt changes in the poly(A) tail length of individual mRNAs or mRNA populations in parallel with changes in their translatability during development (Carrazana et al., 1988; Hyman and Wormington, 1988; Paynton et al., 1988; Robinson et al., 1988; Strickland et al., 1988; Zingg et al., 1988; Carter and Murphy, 1989; McGrew ctal., 1989; Vassalli et al., 1989), Correspondenceto: Dr. A. Jacobson, Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 (U.S.A.) Te!.(508)856-2442; Fax (508) 856-5920. * Current address: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.) Tel. (617)253 3020. Abbreviations: aa, amino acid(s); bp, base pair(s); GI0, uncharacterized
have generated renewed interest in the function of poly(A) and its possible role in gene regulation. Since the discovery of mRNA poly(A) tails almost two decades ago (Kates, 1970; Lim and Caneilakis, 1970; Darnell et al., 1971a,b; Edmonds et el., 1971; Lee et al., 1971) three general models for their function have been proposed. These postulate a role for poly(A) in mRNA processing and transport, mRNA stability, or protein synthesis. In this review we will focus on the evidence which implicates a role for poly(A) in protein synthesis, and in particular, a role in the regulation of mRNA translational efficiency. (See recent reviews by Jacob et al., 1990, and Bernstein and Ross, 1990, for discussions ofpolyadenylation and of tthe possible role of poly(A) in mRNA stability). GENERAL DISCUSSION
Xenopusmaternal mRNA; kb, kilobase(s) or 1000 bp; mRNP, messenger ribonucleoprotein particle; nt, nucleotide(s); PABP, poly(A)-bindingprotein; poly(A) ÷ RNA, RNA containing a Y-poly(A) tract; poly(A)- RNA, RNA lacking a Y-poly(A) tract; r, ribosomal; RNP, ribcnueleoprotein particle; spb, yeast gene encoding suppressors of PABP mutations; tPA, tissue-type plasminogen activator; UTR, untranslated region. 0378-1119/90/$03.50 © 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)
(a) Correlations between the adenylation status of mRNA and its translatability in vivo and in vitro In addition to the studies alluded to above, there are numerous other experimental systems (especially develop-
152 ing systems) in which a direct correlation has been observed between the polyadenylation status ofmRNA and its translational efficiency (i.e., extent of polysome loading) in vivo. Perhaps the most well known of these correlations involves the fate of five developmentally regulated mRNAs upon fertilization of Spisula oocytes (Rosenthal et a1., 1983). Four of these transcripts are poly(A)-mRNAs and are translationally inactive in the oocyte. Following fertilization, these mRNAs becorae adenylated and are simultaneously recruited onto polysomes. In contrast, a fifth transcript, a-tubulin mRNA, is deadenylated at fertilization, coincident with its exclusion from polysomes, Similar examples of the efficient translation of poly(A)+mRNAs and the inefficient translation of poly(A)-mRNAs, and of the interconversion between these states as a consequence of developmental or environmental stimuli, have been observed in species as diverse as Dictyostelium dbcoideum and rats (summarized in Table I). The relationship between poly(A) tail length and translational efficiency in vivo is not absolute. There are examples of translationally inactive polyadenylated mRNAs (Raft, 1980; Rosenthal etal., 1983) as well as examples of mRNAs which appear to lose their poly(A) tracts as they become translationally active (Hruby and Roberts, 1977; latrou and Dixon, 1977; Kleene, 1989). The possibility that 3'-poly(A) tails might play a role in translation has been extensively tested in several in vitro systems. Ira early studies, a relationship between 3'-poty(A) tails and translation was tested and 'ruled out' by experiments which compared the in vitro translational capacity of adenylated and deadenylated forms of various mRNAs. It was generally concluded that artificial deadenylation (Bard
eta!., 1974; Sippel etal., 1974; Soreq etal., !974; Williamson et al., 1974; Spector et al., 1975) or klockage of the poly(A) tail with poly(U) (Munoz and Darnell, 1974) did not significantly reduce the translatability of mRNA. Support for a translational role for poly(A) was subsequevfly resurrected by Doei and Carey (1976) who noted h'mt the translation systems used in the previous studies reinitiated poorly and were thus relatively insensitive to differences in mRNA translational efficiencies. Doel and Carey (1976) were able to show that, while native ovalbumin poly(A)+mRNA was translated more efficiently than its deadenylated counterpart in a highly active reticulocyt,~ extract, no such difference was seen in less active wheat-germ extracts. This translational discrimination was attributed to a reduced rate of initiation on the poly( A ) - m R N A since this mRNA was found on smaller polysomes and participated in fewer rounds of translation. The translational efficiency of mRNAs microinjected into Xenopus oocytes or transferred to plant protoplasts by electroporation also correlates with adenylation status. For example, Deshpande etal. (1979) reported that native ohfglobulin mRNA [average poly(A)~Ts] was translated more efficiently and reached peak translatability faster than poly(A)-poor a2fglobulin mRNA [average poly(A)4o] in microinjected Xenopus oocytes. Drummond et al. (1985) found that synthetic poly(A) + mRNAs, encoding chicken lysozyme, calf preprochymosin, and Xenopus/~-globin were translated 5-20-fold more effectively than poIy(A)-mRNA. Galili et al. (1988) have demonstrated a similar translational discrimination following microinjection of synthetic maize zein or Xenopus/~-globin transcripts into Xenopus oocytes. Furthermore, they reported that the
TABLE 1 Correlations of poly(A)tail length and translationalefficiencyin vivo Organism/conditions
mRNA
Correlation"
Reference
Dlctyostellumdiscoideum/development Urechbcaupo/oogensis
Vegetative and developingmRNAs Maternal mRNAs Total mRNA hsp90 mRNA Different mRNAs
A and B A and B A and B A A and B
Pa]atnik eta]. (1984); Shapiro eta]. (1988) Rosentha] and Wilt (1986) Nemer eta]. (1975) Bedard and Brandhorst (1986) Rosentha] et al. (1983); Rosenthal and Ruderman (1987) Hyman and Wormington (1988) McGrew eta]. (1989) Restifo and Guild (1986)
Sea urchin embryo Sea urchin/oogenesis
Spbula/fertilization
Xenopuslaevbloogenesis r-protein mRNAs Xenopuslaevb/oogenesis GI0 mRNA Drosophila melanogaster/prepupalsalivary Different mRNAs gland develcpment Mouse/oogenesis
B A B
Mouse/oogenesis
Different mRNAs Tissue plasminogenactivator mRNA
A and B A
Rat/circadian rhythm Rat/osmotic stress
Vasopressin mRNA Vasopressin mRNA
A and B A and B
Paynton eta]. (1988) Huarte eta]. (1987); Strickland eta]. (1988); Vassa]li eta]. (1989) Robinson eta]. (1988) Carrazana eta], (1988); Zingg eta]. (1988); Carter and Murphy (1989)
" A, adenylationof mRNA correlates with recruitmentonto polysomes;B, deadenylationof mRNA correlates with exclusionfrom polysomes.
153 poly(A) + mRNAs were found on polysomes with a larger average number of ribosomes than their unadenylated counterparts, indicating that the poly(A)-mRNAs were. deficient in translational initiation. In off,er experiments; synthetic poly(A) + RNA, encoding r-protein L 1, was found to be actively translated following microinjection into stage VI oocytes. Upon oocyte maturation these mRNAs became deadenylated and released from polysomes, paralleling the deadenylation and translational inactivation of endogenous r-protein mRNAs (Hyman and Wormington, 1988). Recently, Gallic etal. (1989) have investigated the translational role of Y-POly(A) tracts in plants. They observed that a synthetic u-glucuronidase (A)~omRNA is translated 16-120-fold more effectively than a similar poly(A)-mRNA in electroporated tobacco, carrot, maize, and rice protoplasts. (b) The cytoplasmic poly(A)-hinding protein (PABP): indirect evidence that it has a role ia translation and that it is a mediator of poly(A) function The mRNAs of every eukaryotic cell are found in ribonucleoprotein complexes (mRNPs), intimately associated with as many as 20 different polypeptide species (Irwin et al., 1975; Jeffery, 1977; Mirkes, 1977; Jain and Sarkar, 1979; Greenberg, 1980; Adams et al., 1981; Setyono and Greenberg, 1981; Moon, 1983; Manrow and Jacobson, 1986; 1987; Bandziulis et al., 1989). By far, the most widely studied of the mRNP proteins are those which associate with the poly(A) tract of mRNA, the PABPs. First described by Blobel (1973), PABPs have been found in every eukaryotic cell examined thus far. Sequence analysis has revealed extensive evolutionary conservation of the yeast, XenoDus, and human PABP-encoding genes (Adam et al., 1986, Sachs et al., 1986; Grange et al., 1987; Zelus et al., 1989). Biochemical analysis of purified yeast PABP has established that: (1) the packing density is approx. 1 PABP per (A)2s (in striking agreement with the size of poly(A) protected from nuclease digestion by PABP; Baer and Kornberg, 1980; 1983); (2) the minimum length of poly(A) required for efficient binding to PABP is 12nt; and (3) PABP can bind to (A)!80_220, and the A-rich domain of the 5'-untranslated region of the PABP mRNA with equal efficiency (Sachs et al., 1986; 1987). Deletion analyses have shown that the PABP is essential for cell viability (Sachs et al., 1986; 1987). Both the evolutionary conservation of the PABP and its necessity for cell viability obviously reflect an important cellular function. Earlier experimental evidence suggested that PABP may mediate the translational effect of 3'-poly(A) tracts. The first such data was provided by Jacobson and Favreau (1983) who reasoned that if 3'-poly(A) tracts did function in protein synthesis then exogenous poly(A) mig,ht competitively inhibit the in vitro translation of poly(A) + mRNAs in
much the same way that 5' -cap structures inhibit the in vitro translation ofcapped mRNAs (Hickey et al., 1976; Roman et al., 1976). They found that exogenous poly(A) inhibited the translation of poly(A)+mRNA, but not poly(A)-mRNA, in reticulocyte lysates and that comparable inhibition was not observed with other polyribonucleotides (Jacobso~l and Favreau, 1983). This poly(A)-mediated inhibition was dependent upon the size of the competitor poly(A) and could be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. Furthermore, poly(A)-mediated inhibition did not alTectthe average size of the polypeptides synthesized, suggesting that this inhibition occurs at the level of translational initiation. These observations have been confirmed by others in reticulocyte lysates (Bablanian and Banerjee, 1986; Grossi de Sa et al., 1988), L-cell lysates (LeMay and Millward, 1986), dry pea-seed extracts (Sieliwanowicz, 1987) and in Xenopusoocytes (Drummond et al., 1985). The most straightforward and generally accepted interpretation of these results is that exogenous poly(A) inhibits translation by fimiting the availability of unbound PABP. Consistent with this interpretation, the addition of purified PABP has been reported to overcome poly(A)-mediated translational inhibition in reticulocyte iysates (Grossi de Sa et al., 1988). These experiments do not, however, exclude the possibility that exogenous poly(A) has a high affinity for another component of the translation apparatus and that addition of purified PABP simply competes for this interaction. Other data supporting a role for the PABP in translation have been provided by Manrow and Jacobson (1986: 1987). They found that, during the first 30 min of DictyosteIium discoideum developm~,~t, when translatic~nal initiation is sharply reduced (Cardelli et al., 1981), the PABPs are selectively degraded (Manrow and Jacobson, 1986). The turnover of the PABPs increased 20-25-fold at this time, resulting in an 80-.90% decrease in their relative abundance. In related experiments (Manrow and Jacobson, 1987), similar reductions in PABP stability and abundance were found to accompany a heat-shock induced reduction in translational initiation. In contrast, the level of PABPs was not affecte~ by reductions in the rate of translational elongation (Manrow and Jacobson, 1987). Additional indirect evidence for a translational role for the PABP has been obtained by Sieliwanowicz (1987) who showed that the translational activity of an in vitro translation system derived from the embryo axes of dry pea seeds is directly related to the relative abundance of the pea 60-kDa PABP. (c) A model for poly(A) function and recent evidence to support it The data discussed thus far, although indirect, are consistent with a translational model for poly(A) function
154
(Jacobsen and Favreau et al., 1983; Palamik ot al., 1984) which postulates that: (l)an interaction between the 3'-poly(A)tract of mRNA and a cytoplasmic PABP specifically enhances translational initiation; (2)this enhancement is directly dependent upon the length of the poly(A) tail such that mRNA with relatively long poly(A) tails has a translational advantage over mRNA with shorter poly(A) tails; (3)this enhancement is not essential for translation; and (4)the regulatory mechanisms which ensure the efficient translation ofpoly(A) + mRNA vs. poly(A)- mRNA may be quite different. Four recent reports (Sachs and Davis, 1989; 1990; Vassalli et al., 1989; Munroe and Jacobsen, 1990) give credence to this model and provide direct evidence for the role played by both poly(A) a~ld PABP in protein synthesis. Those data confu'm thst poly(A) + mRNAs have a translational advantage over poly(A)-mRNAs and establish that: (1) the effect of poly(A) on translation is directly dependent upon its length; (2)the effect of poly(A) on translation is mediated by the PABP; and (3) the poly(A)/PABP complex is involved in translational initiation and, more specifically, in the joining of 60S ribosomal subunits to 48S preinitiation complexes during the formation of 80S initiation complexes. Sachs and Davis (1989) have utilized a genetic approach to demonstrate that the PABP is involved in translational initiation. These experiments involved in vivo depletion of PABP in Saccharomyces cerevisiae by promoter inactivation or by the use of a PABP temperature-sensitive mutation. Depletion of PABP resulted in a marked decrease in the amount of polysomes along with a concomitant increase in free r-subunits, a phenotype characteristic of a translational initiation defect. Seven indepezident, extragenic revertants of the ts mutation which allow translational initiation in the absence of PABP wore isolated. All of these revertants affected cellular levels of the 60S r-subunit. Two revertants (s,vb2-1 and spb4-1) were characterized further and localized, respectively, to the gone for the 60S r-protein L46 and to a gone encoding a putative rRNA helicase involved in the maturation of 25S rRNA (Sachs and Davis, 1989; 1990). Those data indicate that translation in the absence of functional PABP requires an alteration in the structure of the 60S r-subunit. Vassalli et al. (1989) have examined the correlation between polyadonylation and translational ac~vation of ~PA mRNA during meiotic maturation in the mouse oocyte. Microinjection experiments utilizing various tPA chimeric and 3'-blocked in vitro synthesized mRNAs have demonstrated that: (1) this regulated polyadenylation requires sequences present in the 3'-untranslated region of tPA mRNA, and (2) the presence of a long poly(A) tract is both necessary and sufficient for the observed translational recruitment oftPA mRNA. These data show that the trans-
lational discrimination of tPA mRNA before and after germinal vesicle breakdown is due solely to changes in the polyadenylation status of mRNA. Munroe and Jacobsen (1990) have directly compared the translatability of synthetic mRNAs, differing solely in 5'-cap and/or 3'-poly(A) structures, in a cell-free rabbit reticulocyte translation extract. The results of such experiments confirm that poly(A)- mRNAs have a reduced translational capacity and that the effect of poly(A) on translation is directly related to its length. A direct comparison of the polysomes formed by poly(A)+-mRNAs and poly(A)-mRNAs revealed that poly(A)-mRNAs are recruited onto polysomes to a lesser extent than poly(A) + mRNAs and that the polysomes which are formed by poly(A)-mRNAs contain fewer ribosomes than those formed by poly(A) + mRNAs. These data, together with the finding that translational elongation rates along adenylated and unadenylated mRNAs are identical, establish that mRNA poly(A) tails function in translational initiation. Other experiments showed that the defect in poly(A)-mRNAs is distinct from that associated with capdeficient mRNAs and results in a reduced ability to.join 60S r-subunits to 48S preinitiation complexes during the later stage(s) of translational initiation. Utilizing a label transfer protocol, Munro¢ (1989) also showed that the major difference in the mRNP proteins associated with poly(A)+mRNAs or poly(A)-mRNAs is due to proteins which associate with poly(A). Although proteins larger than PABP monomers were detected in these experiments, their Mrs are suggestive of PABP dimers and trimers. The appearance of such putative dimers and trimers increased as a function of poly(A) tail length in a manner consistent with expectatioos based on binding site sizes and in parallel with increases in translational efficiency. These results complement the genetic studies of' Sachs and Davis (1989) and suggest that the functional form of poly(A) is the poly(A)/PABP complex. In other experiments, mRNAs with or without caps and/or poly(A) tails were shown to be differentially sensitive to translational inhibition by exogenous competitor poly(A) (Munroe and Jacobsen, 19901. As expected, the translation of` poly(A) ÷ mRNAs was markedly reduced by the presence of` exogenous poly(A). In contrast, the translation of`capped, unadenylated mRNAs was actually stimulated by exogenous poly(A). No such stimulation was seen with uncapped, unadenylated mRNA implying that: (1) poly(A) may be capable of working in trans to augment the translation of capped, unadenylated mRNAs and (2)event(s) in translational initiation involve the recognition of both 5'.cap and 3'-poly(A) structures (together with their associated proteins). [These observations may also provide a clue to the function of the abundant oligo(A) molecules present amongst the genomic RNAs of
155 reoviruses (Carter et aL, 1974; Stoltzfus et al., 1974). Oligo(A) associated with reovirus particles may act to suppress the translation of capped, host-cell poly(A) ÷ mRNAs while at the same time stimulate the translation of reoviral mRNAs which are normally capped, but poly(A)- ]. Collectively, the experiments of Sachs and Davis (1989; 1990), Munroe (1989), and Munroe and Jacobsen (1990) demonstrate that the poly(A)/PABP complex, and not poly(A) or PABP alone, has a role in the formation of 80S initiation complexes. Such results are consistent with all of the correlative evidence discussed above and also with the observation by Nelson and Winkler (1987) that, in reticulocyte lysates, histone poly(A)- mRNA forms a significantly higher percentage of 'half-mers' (mRNAs bound to 40S r-subunits but lacking 60S r-subunits) than globin poly(A) + mRNA. (d) How is poly(A)/PABP involved in the regulation of gene expression.* Having discussed evidence supporting the involvement of poly(A)/PABP in translational initiation we would now like to speculate about how this complex might function in the regulation of gene expression. Such regulation can be grouped into two broad categories: (l) discrimination between 'old' and 'new' mRNAs, and (2)targeted translational regulation via the manipulation of mRNA adenylation status and/or PABP abundance or structure. mRNA poly(A) tails are heterogeneous in length, ranging, in mammals, from a maximum of (A)25o on newly synthesized mRNA (Brawerman, 1976), to a minimum of about (A)3o (Sheiness and Darnell, 1973; Ahlquist and Kaesberg, 1979). Siace the activity of poly(A) in translation is directly related to its length (Jacobsen and Favreau, 1983; Munroe and Jacobsen, 1990), it would seem logical to predict that newly synthesized mRNA, with relatively long poly(A) tracts, should have a translational advantage over preexisting mRNAs with relatively short poly(A) tracts. Preferential translation of the most recently transcribed sequences would ensure the rapid synthesis of their encoded products. Evidence for translational discrimination between 'new' and 'old' mRNAs has been obtained in Dictyostelium (Palatnik et al., 1984; Shapiro et al., 1988). Likewise, a change in the adenylation status of individual mRNAs or mRNA populations may be sufficient to induce rapid changes in protein synthetic patterns in the absence of significant mRNA synthesis or turnover (Palatnik et al., 1984). As discussed earlier, there are several examples of dramatic changes in translational capacity accompanied by concomitant changes in mRNA adenylation status (Table I). The majority of these changes occur in developing systems, where rapid changes in the pattern of protein synthesis are often necessary. Since such adenylation/ deadenylation may involve only a subset of the cellular
mRNAs (e.g., Sp/sula fertilization; Rosenthal et al., 1983; Rosenthal and Ruderman, 1987), the signals which target individual mRNAs for adenylation and/or deadenylation must be tightly regulated and highly specific. Well-characterized examples of these signals have recently been reported by McGrew et al. (1989) and Vassalli et al. (1989) who identified short, c/s-acting 3'-UTR sequence elements that apparently direct the polyadenylation and subsequent polysomal recruitment of the G 10 and tPA mRNAs during oocyte maturation in Xenopus/aev/s and mice, respectively. The translational effect of poly(A) is almost certainly mediated by the PABP (Sachs and Davis, 1989; 1990; Munroe, 1989). Not surprisingly, changes in the stability and abundance of this protein coincide with changes in the rate of translational initiation which occur during development (Manrow and Jacobsen, 1986) or heat shock (Manrow and Jacobsen, 1987). Similarly, structural variants of the PABP which have retained poly(A)-binding activity, but lost stimulatory activity, may be expected to act as translational repressors. A candidate for such a variant is an oocyte-specific PABP (Swiderski and Richter, 1988) which appears to act as a translational inhibitor (Kick et al., 1987). Thus, the hyper-polyadenylation of'stored' mRNAs following fertilization in Xenopus (McGrew et al., 1989) may occur to provide an accessible binding site for the 'stimulatory form' of PABF. (e) Current models for italy(A) function Verification of the involvement of the poly(A)/PABP complex in translational initiation has led to the proposal of two new models regarding poly(A) function. Sachs and Davis (1989) have proposed that co-recognition of 5'- and 3'-mRNA structures (i.e., caps, poly(A) tails, and their associated proteins) during the initiation of translation may occur simply to prevent the attempted translation of partially degraded mRNAs. On the other hand, Munroe and Jacobson (1990) have proposed that poly(A) sequences be considered translational enhancer sequences which stimulate translational initiation in much the same way that transcriptional enhancers are thought to stimulate the initiation oftranscription (Ptashne, 1986; 1988).These models are not mutually exclusive and both predict that the mRNA 3' and 5' termini interact during translational initiation (Fig. 1). Evidence supporting such an arrangement includes mRNA secondary structure maps which place the mRNA 5' and 3' domains in close proximity (Heindell et al., 1978; Lockard et al., 1986), electron micrographs of polysomes with interacting 5' and 3' ends (Warner etal., 1962; Dubochet etal., 1973; Hsu and Coca-Prados, 1979; Ladhoff eta]., 1981; Christensen etal., 1987), and the demonstration that exogenous poly(A) may be capable of working in trans to stimulate the translation of capped, poly(A)-mRNA (Munroe and Jacobsen, 1990). Con-
156
cap
j
(1) poly(A) is an enhancer ofthe formation of 80S translational initiation complexes; (2) the effect of poly(A) on translation is directly related to its length; (3) the mediator of the function of poly(A) is the cytoplasmic PABP.
ACKNOWLEDGEMENTS Fig. 1. A model for the role of Y-poly(A) tracts in the initiation of translation. We propose that poly(A) acts as the formal equivalent of a transcriptional enhancer. The cartoon depicts the 3'.poly(A): PABP complex promoting the formation of 80S initiation complexes at the mRNA 5'-end (either by a direct interaction with the 60S r-subunit or by facilitating the binding of other protein(s) which, in turn, influence the binding ofthe 60S r-subunit), cap, methylated, inverted nucleotide at the 5' terminus of the mRNA; AAAAA: poly(A) tract at the 3' terminus of the mRNA.
sistent with the enhancer model, is the t'mding that, at low levels, exogenous PABP acts to stimulate in vitro translation of poly(A) + mRNAs while larger amounts exhibit an inhibitory or 'squelching' effect (Sieliwanowicz, 1987). Transcriptional enhancers are known to be orientationindependent. Similarly, the enhancer model for poly(A) function would predict that the location of poly(A) sequences on the mRNA would be unimportant. In this regard, there are several known mRNAs which contain 5'-poly(A) sequences which should be capable of binding PABP, including poxvirus mRNAs (Bertholet et al., 1987; Patel and Pickup, 1987; Schwer et al., 1987) and PABP mRNAs (Adam et al., 1986; Sachs et al., 1986; Grange et al., 1987). The apparent conservation of the 5' A-rich domain between yeast and human PABP mRNAs and amongst the mRNAs of the members of the poxvirus family suggests that it provides some advantage. One possible advantage would be the enhancement of translational initiation even on relatively 'old' mRNAs with shorter 3'-poly(A) tracts. Consideration of the poly(A) tail as a translational enhancer raises the possibility that other sequences in the 3'-UTR also have a translational regulatory role. We anticipate that crosslinkingbstudies and further characterization of the spb mutants ('Sachs and Davis, 1989; 1990) will identify polypeptides which are the targets of PABP function or which stimulate or repress PABP activity. Amongst such polypeptides may be mRNA-binding proteins which, like the PABP, usually bind to sites within the 3'-UTR. (g) Conclusions Recent experiments demonstrate that the 3'-poly(A) tract of eukaryotic mRNAs has a role in translational initiation. These experiments lead us to conclude that:
Work in the authors' laboratory is supported by a grant (GM27757) from the National Institutes of Health. We thank B. Lewin for not letting us down.
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