Possible involvement of poly(A) in protein synthesis.

The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect.     

Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.     

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.     

Characterization of A units released from the poly(A) tract of rabbit globin mRNA during protein synthesis: possible role of the released ATP in synthesizing protein

1. Rabbit globin mRNA poly(A) was translated in two cell-free synthesizing systems, rabbit reticulocyte lysate and wheat germ extract, to characterize the product released from the poly(A) tract during globin synthesis. 2. Kinetic studies indicate that the size of the cleaved nucleotide proves to be a monomer, as revealed by column chromatography on Sephadex G-100 or G-25. 3. Characterization of the monomer was accomplished by chromatography on DEAE-cellulose. Initially, 5 min post-translation, the monomer was ATP only; however, at later times ATP, ADP, AMP and adenosine were detected. 4. The two synthesizing systems differed in that globin mRNA poly(A) was translated at a faster rate in the wheat germ extract as revealed by the appearance of ATP, whereas AMP was detected sooner in the rabbit reticulocyte lysate. 5. The results indicate that the A unit released from the poly(A) tract during mRNA poly(A) translation is a monomer, and that these metabolites may play a role in controlling protein initiation via the released ATP.     

Autoregulation of poly(A)-binding protein synthesis in vitro.

The poly(A)-binding protein (PABP), in a complex with the 3'poly(A) tail of eukaryotic mRNAs, plays important roles in the control of translation and message stability. All known examples of PABP mRNAs contain an extensive A-rich sequence in their 5' untranslated regions. Studies in mammalian cells undergoing growth stimulation or terminal differentiation indicate that PABP expression is regulated at the translational level. Here we examine the hypothesis that synthesis of the PABP is autogenously controlled. We show that the endogenous inactive PABP mRNA in rabbit reticulocytes can be specifically stimulated by addition of low concentrations of poly(A) and that this stimulation is also observed with in vitro transcribed human PABP mRNA. By deleting the A-rich region from the leader of human PABP mRNA and adding it upstream of the initiator AUG in a reporter mRNA we show that the adenylate tract is sufficient and necessary for mRNA repression and poly(A)-mediated activation in the reticulocyte cell-free system. UV cross-linking experiments demonstrate that the leader adenylate tract binds PABP. Furthermore, addition of recombinant GST-PABP to the cell-free system represses translation of mRNAs containing the A-rich sequence in their 5'UTR, but has no effect on control mRNA. We thus conclude that in vitro PABP binding to the A-rich sequence in the 5' UTR of PABP mRNA represses its own synthesis.     

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.     

A direct evidence for the involvement of poly(A) in protein synthesis

A radioactive polyadenylated globin mRNA was translated in either rabbit reticulocyte lysate or wheat germ extract under various conditions. When globin mRNA was translated, globin synthesis was directly proportional to the rate of loss in A units from the poly(A) tail. On the other hand, when globin poly(A) mRNA was incubated under non-translated conditions, no loss of A units was detected. The presence of ribonuclease inhibitor in the reaction mixture did not alter either the rate of globin synthesis or the loss in A units from the poly(A) tail. The present data suggests a correlation between protein synthesis and loss in A units from the poly(A) tail.     

Inhibitory effect of naked neural BC1 RNA or BC200 RNA on eukaryotic in vitro translation systems is reversed by poly(A)-binding protein (PABP)

Regulated protein biosynthesis in dendrites of neurons might be a key mechanism underlying learning and memory. Neuronal dendritic BC1 RNA and BC200 RNA and similar small untranslated RNAs inhibit protein translation in vitro systems, such as rabbit reticulocyte lysate. Likewise, co-transfection of these RNAs with reporter mRNA suppressed translation levels in HeLa cells. The oligo(A)-rich region of all active small RNAs were identified as the RNA domains chiefly responsible for the inhibitory effects. Addition of recombinant human poly(A)-binding protein (PABP) significantly compensated the inhibitory effect of the small oligo(A)-rich RNA. In vivo, all BC1 RNA appears to be complexed with PABP. Nevertheless, in the micro-environment of dendritic spines of neuronal cells, BC1 RNPs or BC200 RNPs might mediate regulatory functions by differential interactions with locally limited PABP and/or directly or indirectly, with other translation initiation factors.     

A 60-kDa protein from rabbit reticulocytes specifically recognizes the capped 5' end of beta-globin mRNA

The binding of proteins from rabbit reticulocyte lysate to in-vitro-generated beta-globin mRNA and its defined segments was investigated using ultraviolet-cross-linking experiments as well as gel-retardation assays. Under stringent conditions, only three proteins (72, 60 and 50 kDa) were found associated with full-length beta-globin mRNA at different positions. The 72-kDa protein is most likely the poly(A)-binding protein and binds, as expected, to the poly(A) tail, whereas the 50-kDa protein exhibits affinity for the trailer region of beta-globin mRNA. The binding region of the 60-kDa protein is located at the 5' end of beta-globin mRNA. The interaction of this protein is dependent on the presence of the 5' cap structure, as indicated by competition experiments using an uncapped beta-globin-mRNA leader segment. Further competition experiments with beta-globin mRNA, deleted in part in the leader region, suggest that, besides the cap structure, certain sequence elements are necessary for the interaction of the 60-kDa protein and the beta-globin mRNA leader.     

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.     

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.     

Translational regulation of human beta interferon mRNA: association of the 3'' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.     

An inhibitor(s) of globin mRNA translation in rabbit serum

1. A factor found in rabbit serum inhibits globin mRNA translation in vitro. 2. Inhibition of globin mRNA translation has been demonstrated in a cell-free rabbit reticulocyte lysate. 3. The inactivation of globin mRNA translation is not attributed to either serum albumin or ribonuclease activities. 4. Dialyzing the inhibitor for 24 hr at 4 degrees C does not result in the diminution of the inhibiting activity. However, the activity of the inhibitor is destroyed by heating to 70-80 degrees C for 5 min or by treatment with trypsin for 2 hr. 5. Ion exchange chromatography points to the inhibitor being a neutral protein, whereas, polyacrylamide gel electrophoresis reveals one major band with mol. wt 43 kDa. 6. The activity of the inhibiting material 3-fold greater in anemic serum than in normal serum. 7. These studies suggest that rabbit serum contains a protein inhibitor that may play a physiological role in regulating protein synthesis in red cells.     

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.     

S-antigen is coded by [poly A+] mRNA from bovine retina

Total RNA, [poly (A)-] mRNA and [poly (A)+] mRNA purified from bovine retina were translated in vitro in a rabbit reticulocyte lysate system. Immunoprecipitation of translation products with antibodies to the retinal S-antigen (a photoreceptor specific protein involved in autoimmune retinal disease) revealed this protein as a 50,000 daltons band comigrating with purified S-antigen. This indicates that the S-antigen is synthesized in the retina and is not a maturation or degradation product of a larger protein. Its messenger RNA is the polyadenylated RNA, as for some other proteins expressed in nervous tissue.     

mRNA with a <20-nt poly(A) tail imparted by the poly(A)-limiting element is translated as efficiently in vivo as long poly(A) mRNA

The poly(A)-limiting element (PLE) is a conserved sequence that restricts the length of the poly(A) tail to <20 nt. This study compared the translation of PLE-containing short poly(A) mRNA expressed in cells with translation in vitro of mRNAs with varying length poly(A) tails. In transfected cells, PLE-containing mRNA had a <20-nt poly(A) and accumulated to a level 20% higher than a matching control without a PLE. It was translated as well as the matching control mRNA with long poly(A) and showed equivalent binding to polysomes. Translation in a HeLa cell cytoplasmic extract was used to examine the impact of the PLE in the context of varying length poly(A) tails. Here the overall translation of +PLE mRNA was less than control mRNA with the same length poly(A), and the PLE did not overcome the effect of a short poly(A) tail. Because poly(A)-binding protein (PABP) is a dominant effector of poly(A)-dependent translation we reasoned excess PABP in our extract might overwhelm PLE regulation of translation. This was confirmed by experiments where PABP was inactivated with poly(rA) or Paip2, and the effect of both treatments was reversed by addition of recombinant PABP. These data indicate that the PLE functionally substitutes for bound PABP to stimulate translation of short poly(A) mRNA.     

Efficient translation of rotavirus mRNA requires simultaneous interaction of NSP3 with the eukaryotic translation initiation factor eIF4G and the mRNA 3' end

In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3'-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3' end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.     

Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation

Translation of most eukaryotic mRNAs involves the synergistic action between the 5′ cap structure and the 3′ poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.