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.     

In vitro deadenylation of mammalian mRNA by a HeLa cell 3'' exonuclease.

We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.     

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.     

Tpa1p is part of an mRNP complex that influences translation termination, mRNA deadenylation, and mRNA turnover in Saccharomyces cerevisiae

In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Delta) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Delta mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5'-->3' pathway or the 3'-->5' nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Delta strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3' untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.     

Kinetic proofreading scanning models for eukaryotic translational initiation: the cap and poly(A) tail dependency of translation

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5' cap and 3' poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5' untranslated region (UTR) from the 5' terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5' UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5' UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5' UTR: the translation of mRNA with structured 5' UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5' UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.     

Base-pairing between untranslated regions facilitates translation of uncapped, nonpolyadenylated viral RNA

Translationally competent mRNAs form a closed loop via interaction of initiation factors with the 5' cap and poly(A) tail. However, many viral mRNAs lack a cap and/or a poly(A) tail. We show that an uncapped, nonpolyadenylated plant viral mRNA forms a closed loop by direct base-pairing (kissing) of a stem loop in the 3' untranslated region (UTR) with a stem loop in the 5' UTR. This allows a sequence in the 3' UTR to confer translation initiation at the 5'-proximal AUG. This base-pairing is also required for replication. Unlike other cap-independent translation mechanisms, the ribosome enters at the 5' end of the mRNA. This remarkably long-distance base-pairing reveals a novel mechanism of cap-independent translation and means by which mRNA UTRs can communicate.     

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.     

Enhancement of IRES-mediated translation of the c-myc and BiP mRNAs by the poly(A) tail is independent of intact eIF4G and PABP

The poly(A) tail at the 3' end of mRNAs enhances 5' cap-dependent translation initiation. We show that it also enhances IRES-directed translation of two cellular mRNAs in vitro and in vivo. The underlying mechanisms, however, differ fundamentally. In contrast to cap-dependent translation, IRES-driven translation continues to be enhanced by the poly(A) tail following proteolytic cleavage of eIF4G. Moreover, the poly(A) tail stimulates IRES-mediated translation even in the presence of PAIP2 or following effective depletion of the poly(A) binding protein (PABP) from HeLa cell extracts. The PABP-eIF4G bridging complex that is critical for cap-dependent translation is thus dispensable for the enhancement of the IRESs by the poly(A) tail. The polyadenylated mRNA translation from cellular IRESs is also profoundly sensitive to eIF4A activity in vitro. These mechanistic and molecular distinctions implicate the potential for a new layer of translational control mechanisms.     

Nucleophosmin deposition during mRNA 3' end processing influences poly(A) tail length

During polyadenylation, the multi-functional protein nucleophosmin (NPM1) is deposited onto all cellular mRNAs analysed to date. Premature termination of poly(A) tail synthesis in the presence of cordycepin abrogates deposition of the protein onto the mRNA, indicating natural termination of poly(A) addition is required for NPM1 binding. NPM1 appears to be a bona fide member of the complex involved in 3' end processing as it is associated with the AAUAAA-binding CPSF factor and can be co-immunoprecipitated with other polyadenylation factors. Furthermore, reduction in the levels of NPM1 results in hyperadenylation of mRNAs, consistent with alterations in poly(A) tail chain termination. Finally, knockdown of NPM1 results in retention of poly(A)(+) RNAs in the cell nucleus, indicating that NPM1 influences mRNA export. Collectively, these data suggest that NPM1 has an important role in poly(A) tail length determination and may help network 3' end processing with other aspects of nuclear mRNA maturation.     

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.     

Conventional 3' end formation is not required for NMD substrate recognition in Saccharomyces cerevisiae

The recognition and rapid degradation of mRNAs with premature translation termination codons by the nonsense-mediated pathway of mRNA decay is an important RNA quality control system in eukaryotes. In mammals, the efficient recognition of these mRNAs is dependent upon exon junction complex proteins deposited on the RNA during pre-mRNA splicing. In yeast, splicing does not play a role in recognition of mRNAs that terminate translation prematurely, raising the possibility that proteins deposited during alternative pre-mRNA processing events such as 3' end formation might contribute to the distinction between normal and premature translation termination. We have utilized mRNAs with a 3' poly(A) tail generated by ribozyme cleavage to demonstrate that the normal process of 3' end cleavage and polyadenylation is not required for mRNA stability or the detection of a premature stop codon. Thus, in yeast, the distinction between normal and premature translation termination events is independent of both splicing and conventional 3' end formation.     

Identification of two cis-acting elements that independently regulate the length of poly(A) on Xenopus albumin pre-mRNA.

Unlike most eukaryotic mRNAs studied to date, Xenopus serum albumin mRNA has a short (17-residue), discrete poly(A) tail. We recently reported that this short poly(A) tail results from regulation of the length of poly(A) on albumin pre-mRNA. The purpose of the present study was to locate the cis-acting element responsible for this, the poly(A)-limiting element or PLE. An albumin minigene consisting of albumin cDNA joined in exon 13 to the 3' end of the albumin gene produced mRNA with <20 nt poly(A) when transfected into mouse fibroblasts. This result indicates both that cis-acting sequences that regulate poly(A) length are within this construct, and that nuclear regulation of poly(A) length is conserved between vertebrates. Poly(A) length regulation was retained after replacing the terminal 53 bp and 3' flanking region of the albumin gene with a synthetic polyadenylation element (SPA). Conversely, fusing albumin gene sequence spanning the terminal 53 bp of the albumin gene and 3' flanking sequence onto the human beta-globin gene yielded globin mRNA with a 200-residue poly(A)tail. These data indicate that the PLE resides upstream of the sequence elements involved in albumin pre-mRNA 3' processing. Poly(A) length regulation was restored upon fusing a segment bearing albumin intron 14, exon 15, and 3' flanking sequence onto the beta-globin gene. We demonstrate that exon 15 contains two PLEs that can act independently to regulate the length of poly(A).     

Assaying the polyadenylation state of mRNAs

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.     

Structure and function of a cap-independent translation element that functions in either the 3' or the 5' untranslated region

Barley yellow dwarf virus RNA lacks both a 5' cap and a poly(A) tail, yet it is translated efficiently. It contains a cap-independent translation element (TE), located in the 3' UTR, that confers efficient translation initiation at the AUG closest to the 5' end of the mRNA. We propose that the TE must both recruit ribosomes and facilitate 3'-5' communication. To dissect its function, we determined the secondary structure of the TE and roles of domains within it. Nuclease probing and structure-directed mutagenesis revealed that the 105-nt TE (TE105) forms a cruciform secondary structure containing four helices connected by single-stranded regions. TE105 can function in either UTR in wheat germ translation extracts. A longer viral sequence (at most 869 nt) is required for full cap-independent translation in plant cells. However, substantial translation of uncapped mRNAs can be obtained in plant cells with TE105 combined with a poly(A) tail. All secondary structural elements and most primary sequences that were mutated are required for cap-independent translation in the 3' and 5' UTR contexts. A seven-base loop sequence was needed only in the 3' UTR context. Thus, this loop sequence may be involved only in communication between the UTRs and not directly in recruiting translational machinery. This structural and functional analysis provides a framework for understanding an emerging class of cap-independent translation elements distinguished by their location in the 3' UTR.     

A four-nucleotide translation enhancer in the 3'-terminal consensus sequence of the nonpolyadenylated mRNAs of rotavirus

The 5' cap and poly(A) tail of eukaryotic mRNAs work synergistically to enhance translation through a process that requires interaction of the cap-associated eukaryotic initiation factor, eIF-4G, and the poly(A)-binding protein, PABP. Because the mRNAs of rotavirus, and other members of the Reoviridae, contain caps but lack poly(A) tails, their translation may be enhanced through a unique mechanism. To identify translation-enhancement elements in the viral mRNAs that stimulate translation in vivo, chimeric RNAs were prepared that contained an open reading frame for luciferase and the 5' and 3' untranslated regions (UTRs) of a rotavirus mRNA or of a nonviral mRNA. Transfection of the chimeric RNAs into rotavirus-infected cells showed that the viral 3' UTR contained a translation-enhancement element that promoted gene expression. The element did not enhance gene expression in uninfected cells and did not affect the stability of the RNAs. Mutagenesis showed that the conserved sequence GACC located at the 3' end of rotavirus mRNAs operated as an enhancement element. The 3'-GACC element stimulated protein expression independently of the sequence of the 5' UTR, although efficient expression required the RNA to contain a cap. The results indicate that the expression of viral proteins in rotavirus-infected cells is specifically up-regulated by the activity of a novel 4-nt 3' translation enhancer (TE) common to the 11 nonpolyadenylated mRNAs of the virus. The 4-nt sequence of the rotavirus 3' TE represents by far the shortest of any of the sequence enhancers known to stimulate translation.     

Control of translation by the 5'- and 3'-terminal regions of the dengue virus genome

The genomic RNAs of flaviviruses such as dengue virus (DEN) have a 5' m7GpppN cap like those of cellular mRNAs but lack a 3' poly(A) tail. We have studied the contributions to translational expression of 5'- and 3'-terminal regions of the DEN serotype 2 genome by using luciferase reporter mRNAs transfected into Vero cells. DCLD RNA contained the entire DEN 5' and 3' untranslated regions (UTRs), as well as the first 36 codons of the capsid coding region fused to the luciferase reporter gene. Capped DCLD RNA was as efficiently translated in Vero cells as capped GLGpA RNA, a reporter with UTRs from the highly expressed alpha-globin mRNA and a 72-residue poly(A) tail. Analogous reporter RNAs with regulatory sequences from West Nile and Sindbis viruses were also strongly expressed. Although capped DCLD RNA was expressed much more efficiently than its uncapped form, uncapped DCLD RNA was translated 6 to 12 times more efficiently than uncapped RNAs with UTRs from globin mRNA. The 5' cap and DEN 3' UTR were the main sources of the translational efficiency of DCLD RNA, and they acted synergistically in enhancing translation. The DEN 3' UTR increased mRNA stability, although this effect was considerably weaker than the enhancement of translational efficiency. The DEN 3' UTR thus has translational regulatory properties similar to those of a poly(A) tail. Its translation-enhancing effect was observed for RNAs with globin or DEN 5' sequences, indicating no codependency between viral 5' and 3' sequences. Deletion studies showed that translational enhancement provided by the DEN 3' UTR is attributable to the cumulative contributions of several conserved elements, as well as a nonconserved domain adjacent to the stop codon. One of the conserved elements was the conserved sequence (CS) CS1 that is complementary to cCS1 present in the 5' end of the DEN polyprotein open reading frame. Complementarity between CS1 and cCS1 was not required for efficient translation.     

How strong is the case for regulation of the initiation step of translation by elements at the 3' end of eukaryotic mRNAs?

The belief that initiation of translation requires communication between the 5' and 3' ends of the mRNA guides--or misguides--the interpretation of many experiments. The closed-loop model for initiation creates the expectation that sequences at the 3' end of eukaryotic mRNAs should regulate translation. This review looks closely at the evidence in three prominent cases where such regulation is claimed. The mRNAs in question encode 15-lipoxygenase, ceruloplasmin, and histones. Vertebrate histone mRNAs lack a poly(A) tail, instead of which a 3' stem-loop structure is said to promote translation by binding a protein which purportedly binds initiation factors. The proffered evidence for this hypothesis has many flaws. Temporal control of 15-lipoxygenase production in reticulocytes is often cited as another well-documented example of translational regulation via the 3' untranslated region, but inspection of the evidence reveals significant gaps and contradictions. Solid evidence is lacking also for the idea that a ribosomal protein binds to and shuts off translation of ceruloplasmin mRNA. Some viral RNAs that lack a poly(A) tail have alternative 3' structures which are said to promote translation via circularization of the mRNA, but in no case has this been shown convincingly. Interpretation of many experiments is compromised by possible effects of the 3' structures on mRNA stability rather than translation. The functional-half-life assay, which is often employed to rule out effects on mRNA stability, might not be adequate to settle the question. Other issues, such as the possibility of artifacts caused by overexpression of RNA-binding proteins, can complicate studies of translational regulation. There is no doubt that elements at the 3' end of eukaryotic mRNAs can regulate gene expression in a variety of ways. It has not been shown unequivocally that one of these ways involves direct participation of the 3' untranslated region in the initiation step of translation.     

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.