In the beginning is the end: regulation of poly(A) addition and removal during early development

The addition of poly(A) tails to nearly all mRNAs within the nucleus was reviewed in the July issue of TIBS. Here we shift focus to the fate of poly(A) tails during early development. At specific times during oogenesis and embryogenesis, the poly(A) tails of certain maternal mRNAs are lengthened, while the tails of a number of other mRNAs are removed. The selective poly(A) addition reactions are regulated by a short, U-rich sequence in the 3' untranslated region, while the removal of poly(A) from specific mRNAs is a 'default state', requiring no specific sequence. These regulated changes in poly(A) length are likely to play a major role in translational regulation in the egg and early embryo.     

Free poly(A) stimulates capped mRNA translation in vitro through the eIF4G-poly(A)-binding protein interaction

The 5' cap and 3' poly(A) tail of classical eukaryotic mRNAs functionally communicate to synergistically enhance translation initiation. Synergy has been proposed to result in part from facilitated ribosome recapture on circularized mRNAs. Here, we demonstrate that this is not the case. In poly(A)-dependent, ribosome-depleted rabbit reticulocyte lysates, the addition of exogenous poly(A) chains of physiological length dramatically stimulated translation of a capped, nonpolyadenylated mRNA. When the poly(A):RNA ratio approached 1, exogenous poly(A) stimulated translation to the same extent as the presence of a poly(A) tail at the mRNA 3' end. In addition, exogenous poly(A) significantly improved translation of capped mRNAs carrying short poly(A(50)) tails. Trans stimulation of translation by poly(A) required the eIF4G-poly(A)-binding protein interaction and resulted in increased affinity of eIF4E for the mRNA cap, exactly as we recently described for cap-poly(A) synergy. These results formally demonstrate that mRNA circularization per se is not the cause of cap-poly(A) synergy at least in vitro.     

Poly(A) dependent translation in rabbit reticulocyte lysate

Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)⁺ and poly(A)⁻ mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease uncreated reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs. stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.     

The sequence 5'-AAUAAA-3'forms parts of the recognition site for polyadenylation of late SV40 mRNAs

We have observed three effects of deletion mutations on polyadenylation of late SV40 mRNAs. The first class of mutants lack segments (-3 to -14 bp) between the 5-AAUAAA-3' and normal poly(A) site. These mutants produce mRNas polyadenylated at new sites, downstream from the wild-type site. The poly(A) site is moved farther downstream as the deletions become larger; as a result, polyadenylation always occurs within an 11-19 nucleotide range from the AAUAAA sequence. The second class of mutants lack segments (-12 to -30 bp) between the AAUAAA sequence and the coding region of the mRNA. The poly(A) site for only one of these mutants was studied (dl1457, -12 bp). In this case, the spatial relationship between AAUAAA and poly(A) site is altered. dl1457 produces a class of mRNAs polyadenylated at the first Ca following the AAUAAA sequence, as well as other mRNAs polyadenylated farther downstream. Finally, a 16 bp deletion that includes the AAUAAA sequence prevents poly(A) addition.     

The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro.

Using an in vitro mRNA decay system, we investigated how poly(A) and its associated poly(A)-binding protein (PABP) affect mRNA stability. Cell extracts used in the decay reactions were depleted of functional PABP either by adding excess poly(A) competitor or by passing the extracts over a poly(A)-Sepharose column. Polyadenylated mRNAs for beta-globin, chloramphenicol acetyltransferase, and simian virus 40 virion proteins were degraded 3 to 10 times faster in reactions lacking PABP than in those containing excess PABP. The addition of purified Saccharomyces cerevisiae or human cytoplasmic PABP to PABP-depleted reactions stabilized the polyadenylated mRNAs. In contrast, the decay rates of nonpolyadenylated mRNAs were unaffected by PABP, indicating that both the poly(A) and its binding protein were required for maintaining mRNA stability. A nonspecific single-stranded binding protein from Escherichia coli did not restore stability to polyadenylated mRNA, and the stabilizing effect of PABP was inhibited by anti-PABP antibody. The poly(A) tract was the first mRNA segment to be degraded in PABP-depleted reactions, confirming that the poly(A)-PABP complex was protecting the 3' region from nucleolytic attack. These results indicate that an important function of poly(A), in conjunction with its binding protein, is to protect polyadenylated mRNAs from indiscriminate destruction by cellular nucleases. A model is proposed to explain how the stability of an mRNA could be affected by the stability of its poly(A)-PABP complex.     

Plant mitochondrial polyadenylated mRNAs are degraded by a 3'- to 5'-exoribonuclease activity, which proceeds unimpeded by stable secondary structures

Recently, we and others have reported that mRNAs may be polyadenylated in plant mitochondria, and that polyadenylation accelerates the degradation rate of mRNAs. To further characterize the molecular mechanisms involved in plant mitochondrial mRNA degradation, we have analyzed the polyadenylation and degradation processes of potato atp9 mRNAs. The overall majority of polyadenylation sites of potato atp9 mRNAs is located at or in the vicinity of their mature 3'-extremities. We show that a 3'- to 5'-exoribonuclease activity is responsible for the preferential degradation of polyadenylated mRNAs as compared with non-polyadenylated mRNAs, and that 20-30 adenosine residues constitute the optimal poly(A) tail size for inducing degradation of RNA substrates in vitro. The addition of as few as seven non-adenosine nucleotides 3' to the poly(A) tail is sufficient to almost completely inhibit the in vitro degradation of the RNA substrate. Interestingly, the exoribonuclease activity proceeds unimpeded by stable secondary structures present in RNA substrates. From these results, we propose that in plant mitochondria, poly(A) tails added at the 3' ends of mRNAs promote an efficient 3'- to 5'- degradation process.     

The Role of the poly(A) sequence in mammalian messenger RNA

The poly(A) sequence is added to 3' termini of nuclear RNA segments destined to become part of the mRNA, and may play an essential role in the selection of these segments. It appears to be required for at least some of the splicing events involved in mRNA processing. In the cytoplasm, the poly(A) segment is the target of a degradation process which causes its gradual shortening, and leads to a heterogeneous steady-state poly(A)-size distribution. Complete loss of the poly(A) is probably followed by inactivation of the mRNA, since chains depleted of poly(A) do not accumulate in the cells. A role for this sequence in the promotion of mRNA stability is suggested by the behavior of globin mRNA depleted of poly(A) after injection into frog oocytes. The poly(A) shortening process may be part of the mRNA inactivation mechanism, as indicated by the greater sensitivity to degradation of the poly(A) of some short-lived mRNAs. However, the stochastic mRNA decay implies that new and old mRNA chains, with long and short poly(A) segments, respectively are equally susceptible to inactivation. The poly(A)-lacking histone mRNAs are stable only in cells engaged in DNA replication. Present knowledge favors a role for poly(A) in the control of mRNA stability. Loss of this sequence could be controlled through modulation of poly(A)-protein interactions or through masking of a sequence directly adjacent to the poly(A). In the nucleus, the poly(A) sequence could also serve as stabilizing agent, but, in addition, it might interact with the splicing machinery.     

Localization and sequence analysis of poly(A) sites generating multiple dihydrofolate reductase mRNAs

The murine dihydrofolate reductase (DHFR) gene gives rise to multiple polyadenylated mRNAs displaying heterogeneity in the length of the 3' untranslated region. These species are present in the cytoplasm at levels that vary over 2 orders of magnitude, suggesting that certain poly(A) sites are preferred over others. Previous observations have shown that three out of the four major sites of polyadenylation do not display consensus hexanucleotide (AATAAA, ATTAAA) signals. We have further analyzed the sequences involved in directing multiple polyadenylation events on the DHFR gene by focusing our attention on the 4.1- and 5.6-kilobase mRNAs, the lowest abundance DHFR species observed on RNA blot analysis. Identification and sequence analysis of the poly(A) addition sites corresponding to these species revealed appropriately positioned consensus hexanucleotide signals; additional nearby poly(A) sites were also detected which apparently do not use consensus hexanucleotides to direct poly(A) addition to DHFR mRNAs of relatively lower abundance. We have also identified polyadenylation sites downstream of the 4.1- and 5.6-kilobase sites which display consensus hexanucleotide signals and correspond to messenger species too rare for detection by routine RNA blot analysis. Our data bring to 11 the number of known functional poly(A) addition sites associated with the DHFR gene.     

Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.     

Mitochondrial Dysfunction Reveals the Role of mRNA Poly(A) Tail Regulation in Oculopharyngeal Muscular Dystrophy Pathogenesis

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.     

Messenger RNA stability in Saccharomyces cerevisiae: the influence of translation and poly(A) tail length.

A comparison between the half-lives of 10 specific yeast mRNAs and their distribution within polysomes (fractionated on sucrose density gradients) was used to test the relationship between mRNA translation and degradation in the eukaryote Saccharomyces cerevisiae. Although the mRNAs vary in their distribution across the same polysome gradients, there is no obvious correlation between the stability of an mRNA and the number of ribosomes it carries in vivo. This suggests that ribosomal protection against nucleolytic attack is not a major factor in determining the stability of an mRNA in yeast. The relative lengths of the poly(A) tails of 9 yeast mRNAs were analysed using thermal elution from poly(U)-Sepharose. No dramatic differences in poly(A) tail length were observed amongst the mRNAs which could account for their wide ranging half-lives. Minor differences were consistent with shortening of the poly(A) tail as an mRNA ages.     

mRNA poly(A) tail, a 3'' enhancer of translational initiation.

To evaluate the hypothesis that the 3' poly(A) tract of mRNA plays a role in translational initiation, we constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs were translated, degraded, recruited into polysomes, and assembled into messenger ribonucleoproteins or intermediates in the translational initiation pathway. Relative to mRNAs which were polyadenylated, we found that nonpolyadenylated [poly(A)-]mRNAs had a reduced translational capacity which was not due to an increase in their decay rates, but was attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A)- mRNAs affected a late step in translational initiation, was distinct from the phenotype associated with cap-deficient mRNAs, and resulted in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibited translation from capped polyadenylated mRNAs but stimulated translation from capped poly(A)- mRNAs. We suggest that the presence of a 3' poly(A) tail may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5' end.