Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro.

In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm. We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments. Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo. RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing. We show that Pab1 and CF I copurify in anion-exchange chromatography. These data suggest that Pab1 is physically associated with CF I. Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length. A normal tail length was restored by adding recombinant Pab1 to the mutant extract. The longer poly(A) tails were not due to an inhibition of exonuclease activities. Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability. Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.     

RNA Recognition Motif 2 of Yeast Pab1p Is Required for Its Functional Interaction with Eukaryotic Translation Initiation Factor 4G

The eukaryotic mRNA 3′ poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.     

Tethering of Poly(A)-binding Protein Interferes with Non-translated mRNA Decay from the 5′ End in Yeast

The decay of eukaryotic mRNA is triggered mainly by deadenylation, which leads to decapping and degradation from the 5′ end of an mRNA. Poly(A)-binding protein has been proposed to inhibit the decapping process and to stabilize mRNA by blocking the recruitment of mRNA to the P-bodies where mRNA degradation takes place after stimulation of translation initiation. In contrast, several lines of evidence show that poly(A)-binding protein (Pab1p) has distinct functions in mRNA decay and translation in yeast. To address the translation-independent function of Pab1p in inhibition of decapping, we examined the contribution of Pab1p to the stability of non-translated mRNAs, an AUG codon-less mRNA or an mRNA containing a stable stem-loop structure at the 5′-UTR. Tethering of Pab1p stabilized non-translated mRNAs, and this stabilization did not require either the eIF4G-interacting domain of Pab1p or the Pab1p-interacting domain of eIF4G. In a ski2Δ mutant in which 3′ to 5′ mRNA degradation activity is defective, stabilization of non-translated mRNAs by the tethering of Pab1p lacking an eIF4G-interacting domain (Pab1–34Cp) requires a cap structure but not a poly(A) tail. In wild type cells, stabilization of non-translated mRNA by tethered Pab1–34Cp results in the accumulation of deadenylated mRNA. These results strongly suggest that tethering of Pab1p may inhibit the decapping reaction after deadenylation, independent of translation. We propose that Pab1p inhibits the decapping reaction in a translation-independent manner in vivo.     

Secondary structure in the 5'-leader or 3'-untranslated region reduces protein yield but does not affect the functional interaction between the 5'-cap and the poly(A) tail

The 5'-cap structure and poly(A) tail of eukaryotic mRNAs cooperate to promote translation initiation but whether this functional interaction benefits certain classes of mRNAs has not been investigated. In this study, we investigate whether a structured 5'-leader or 3'-untranslated region (UTR) affects the cap/poly(A) tail interaction. A structured leader reduced the degree to which the 5'-cap promoted translation in plant cells and inhibited translation from capped and uncapped mRNAs equally in yeast. Secondary structure within the 3'-UTR reduced translational efficiency when adjacent to the stop codon but had little effect on the cap/poly(A) tail synergy. The functional interaction between the cap and poly(A) tail was as important for an mRNA with a structured leader or 3'-UTR as it was for an unstructured mRNA in either species, suggesting that these structures can reduce translation without affecting the functional interaction between the cap and poly(A) tail. However, the loss of Xrn1p, the major 5'-->3' exoribonuclease in yeast, abolished cap-dependent translation and the functional interaction between the cap and poly(A) tail, suggesting that the cap/poly(A) tail synergy is of particular importance under conditions of active RNA turnover.     

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.     

Molecular dissection of mRNA poly(A) tail length control in yeast

In eukaryotic cells, newly synthesized mRNAs acquire a poly(A) tail that plays several fundamental roles in export, translation and mRNA decay. In mammals, PABPN1 controls the processivity of polyadenylation and the length of poly(A) tails during de novo synthesis. This regulation is less well-detailed in yeast. We have recently demonstrated that Nab2p is necessary and sufficient for the regulation of polyadenylation and that the Pab1p/PAN complex may act at a later stage in mRNA metabolism. Here, we show that the presence of both Pab1p and Nab2p in reconstituted pre-mRNA 3′-end processing reactions has no stimulating nor inhibitory effect on poly(A) tail regulation. Importantly, the poly(A)-binding proteins are essential to protect the mature mRNA from being subjected to a second round of processing. We have determined which domains of Nab2p are important to control polyadenylation and found that the RGG-box work in conjunction with the two last essential CCCH-type zinc finger domains. Finally, we have tried to delineate the mechanism by which Nab2p performs its regulation function during polyadenylation: it likely forms a complex with poly(A) tails different from a simple linear deposit of proteins as it has been observed with Pab1p.     

Multiple portions of poly(A)-binding protein stimulate translation in vivo

Translational stimulation of mRNAs during early development is often accompanied by increases in poly(A) tail length. Poly(A)-binding protein (PAB) is an evolutionarily conserved protein that binds to the poly(A) tails of eukaryotic mRNAs. We examined PAB's role in living cells, using both Xenopus laevis oocytes and Saccharomyces cerevisiae, by tethering it to the 3'-untranslated region of reporter mRNAs. Tethered PAB stimulates translation in vivo. Neither a poly(A) tail nor PAB's poly(A)-binding activity is required. Multiple domains of PAB act redundantly in oocytes to stimulate translation: the interaction of RNA recognition motifs (RRMs) 1 and 2 with eukaryotic initiation factor-4G correlates with translational stimulation. Interaction with Paip-1 is insufficient for stimulation. RRMs 3 and 4 also stimulate, but bind neither factor. The regions of tethered PAB required in yeast to stimulate translation and stabilize mRNAs differ, implying that the two functions are distinct. Our results establish that oocytes contain the machinery necessary to support PAB-mediated translation and suggest that PAB may be an important participant in translational regulation during early development.     

Nonsense-mediated mRNA decay in yeast does not require PAB1 or a poly(A) tail

Eukaryotic mRNAs harboring premature translation termination codons are recognized and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. The mechanism for discriminating between mRNAs that terminate translation prematurely and those subject to termination at natural stop codons remains unclear. Studies in multiple organisms indicate that proximity of the termination codon to the 3' poly(A) tail and the poly(A) RNA-binding protein, PAB1, constitute the critical determinant in NMD substrate recognition. We demonstrate that mRNA in yeast lacking a poly(A) tail can be destabilized by introduction of a premature termination codon and, importantly, that this mRNA is a substrate of the NMD machinery. We further show that, in cells lacking Pab1p, mRNA substrate recognition and destabilization by NMD are intact. These results establish that neither the poly(A) tail nor PAB1 is required in yeast for discrimination of nonsense-codon-containing mRNA from normal by NMD.     

Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export

Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3'-end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)-binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A)(+) RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail-binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2Delta hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export.     

The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex

Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation. The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G. Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo. These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled.     

The role of 5'-leader length, secondary structure and PABP concentration on cap and poly(A) tail function during translation in Xenopus oocytes

The 5′-cap structure and poly(A) tail of eukaryotic mRNAs function synergistically to promote translation initiation through a physical interaction between the proteins that bind to these regulatory elements. In this study, we have examined the effect of leader length and the presence of secondary structure on the translational competence and the function of the cap and poly(A) tail for mRNAs microinjected into Xenopus oocytes. Increasing the length of the 5′-leader from 17 to 144 nt resulted in a 2- to 4-fold increase in expression from an mRNA containing an unstructured leader but increased expression up to 20-fold for an mRNA containing 5′-proximal structure. Consequently, the presence of secondary structure was less inhibitory for those mRNAs with a longer 5′-leader. Co-injection of poly(A)-binding protein (PABP) mRNA increased the function of the cap and poly(A) tail in promoting translation from poly(A)⁺ but not poly(A)^– mRNAs, particularly for mRNAs containing secondary structure. In the absence of an internal ribosome entry site, expression from the distal cistron of a dicistronic mRNA increased as a function of the length of the intercistronic region and the concentration of PABP. The inhibitory effect of intercistronic located secondary structure on translation was position-dependent. Indeed, the effect of secondary structure was abolished if positioned 134 nt upstream of the distal cistron. These data suggest that the length of a leader, the presence of secondary structure and the concentration of PABP determine the extent to which the cap and poly(A) tail regulate translation.     

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.     

Yeast mRNA Poly(A) tail length control can be reconstituted in vitro in the absence of Pab1p-dependent Poly(A) nuclease activity

Regulation of poly(A) tail length during mRNA 3'-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3'-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3'-end formation were purified from wild-type and pan mutant cells. In both situations, 3'-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast.     

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.     

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.     

Regulation of the nuclear poly(A)-binding protein by arginine methylation in fission yeast

Two structurally different poly(A)-binding proteins (PABP) bind the poly(A) tract of mRNAs in most mammalian cells: PABPC in the cytoplasm and PABP2/PABPN1 in the nucleus. Whereas yeast orthologs of the cytoplasmic PABP are characterized, a gene product homologous to mammalian PABP2 has not been identified in yeast. We report here the identification of a homolog of PABP2 as an arginine methyltransferase 1 (RMT1)-associated protein in fission yeast. The product of the Schizosaccharomyces pombe pab2 gene encodes a nonessential nuclear protein and demonstrates specific poly(A) binding in vitro. Consistent with a functional role in poly(A) tail metabolism, mRNAs from pab2-null cells displayed hyperadenylated 3'-ends. We also show that arginine residues within the C-terminal arginine-rich domain of Pab2 are modified by RMT1-dependent methylation. Whereas the arginine methylated and unmethylated forms of Pab2 behaved similarly in terms of subcellular localization, poly(A) binding, and poly(A) tail length control; Pab2 oligomerization levels were markedly increased when Pab2 was not methylated. Significantly, Pab2 overexpression reduced growth rate, and this growth inhibitory effect was exacerbated in rmt1-null cells. Our results indicate that the main cellular function of Pab2 is in poly(A) tail length control and support a biological role for arginine methylation in the regulation of Pab2 oligomerization.     

Linking the 3′ Poly(A) Tail to the Subunit Joining Step of Translation Initiation: Relations of Pab1p, Eukaryotic Translation Initiation Factor 5B (Fun12p), and Ski2p-Slh1p

The 3' poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A)(+) mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A)(+) mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining.