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.     

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.     

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.     

Analysis of an essential requirement for the poly(A) binding protein function using cross-species complementation

Poly(A) binding protein (PABP) is an essential, well-conserved, multifunctional protein involved in translational initiation, mRNA biogenesis, and degradation [1--5]. We have used a cross-species complementation approach to address the nature of the essential requirement for PABP in yeast. The expression of Pab3p, a member of the Arabidopsis thaliana PABP multigene family, rescues the lethal phenotype associated with the loss of the yeast Pab1p. However, Pab3p neither protects the mRNA 5' cap from premature removal, nor does it support poly(A)-dependent translational initiation or the synergistic enhancement of translation by the poly(A) tail and 5' cap in yeast. However, Pab3p corrects the temporal lag prior to the entry of the mRNA into the degradation pathway characteristic of pab1 Delta yeast strains. Furthermore, this lag correction by Pab3p requires Pan3p, a subunit of poly(A) nuclease, an enzyme involved in the mRNA 3'-end processing. Importantly, the substitution of Pab3p for the yeast Pab1p is synthetically lethal with the PAN3 gene deletion. These results show that the function of PABP in mRNA biogenesis alone could be sufficient to support cell viability in yeast.     

Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins

Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here we find that the nuclear exosome subunit Rrp6p counteracts the in vitro and in vivo extension of mature pA tails by the noncanonical pA polymerase Trf4p. Moreover, PABP loading onto nascent pA tails is controlled by Rrp6p; while Pab1p is the major PABP, Nab2p only associates in the absence of Rrp6p. This is because Rrp6p can interact with Nab2p and displace it from pA tails, potentially leading to RNA turnover, as evidenced for certain pre-mRNAs. We suggest that a nuclear mRNP surveillance step involves targeting of Rrp6p by Nab2p-bound pA-tailed RNPs and that pre-mRNA abundance is regulated at this level.     

Evidence that poly(A) binding protein has an evolutionarily conserved function in facilitating mRNA biogenesis and export

Eukaryotic poly(A) binding protein (PABP) is a ubiquitous, essential cellular factor with well-characterized roles in translational initiation and mRNA turnover. In addition, there exists genetic and biochemical evidence that PABP has an important nuclear function. Expression of PABP from Arabidopsis thaliana, PAB3, rescues an otherwise lethal phenotype of the yeast pab1Delta mutant, but it neither restores the poly(A) dependent stimulation of translation, nor protects the mRNA 5' cap from premature removal. In contrast, the plant PABP partially corrects the temporal lag that occurs prior to the entry of mRNA into the decay pathway in the yeast strains lacking Pab1p. Here, we examine the nature of this lag-correction function. We show that PABP (both PAB3 and the endogenous yeast Pab1p) act on the target mRNA via physically binding to it, to effect the lag correction. Furthermore, substituting PAB3 for the yeast Pab1p caused synthetic lethality with rna15-2 and gle2-1, alleles of the genes that encode a component of the nuclear pre-mRNA cleavage factor I, and a factor associated with the nuclear pore complex, respectively. PAB3 was present physically in the nucleus in the complemented yeast strain and was able to partially restore the poly(A) tail length control during polyadenylation in vitro, in a poly(A) nuclease (PAN)-dependent manner. Importantly, PAB3 in yeast also promoted the rate of entry of mRNA into the translated pool, rescued the conditional lethality, and alleviated the mRNA export defect of the nab2-1 mutant when overexpressed. We propose that eukaryotic PABPs have an evolutionarily conserved function in facilitating mRNA biogenesis and export.     

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.     

Pbp1p, a Factor Interacting with Saccharomyces cerevisiae Poly(A)-Binding Protein, Regulates Polyadenylation

The poly(A) tail of an mRNA is believed to influence the initiation of translation, and the rate at which the poly(A) tail is removed is thought to determine how fast an mRNA is degraded. One key factor associated with this 3′-end structure is the poly(A)-binding protein (Pab1p) encoded by the PAB1 gene in Saccharomyces cerevisiae. In an effort to learn more about the functional role of this protein, we used a two-hybrid screen to determine the factor(s) with which it interacts. We identified five genes encoding factors that specifically interact with the carboxy terminus of Pab1p. Of a total of 44 specific clones identified, PBP1 (for Pab1p-binding protein) was isolated 38 times. Of the putative interacting genes examined, PBP1 promoted the highest level of resistance to 3-aminotriazole (>100 mM) in constructs in which HIS3 was used as a reporter. We determined that a fraction of Pbp1p cosediments with polysomes in sucrose gradients and that its distribution is very similar to that of Pab1p. Disruption of PBP1 showed that it is not essential for viability but can suppress the lethality associated with a PAB1 deletion. The suppression of pab1Δ by pbp1Δ appears to be different from that mediated by other pab1 suppressors, since disruption of PBP1 does not alter translation rates, affect accumulation of ribosomal subunits, change mRNA poly(A) tail lengths, or result in a defect in mRNA decay. Rather, Pbp1p appears to function in the nucleus to promote proper polyadenylation. In the absence of Pbp1p, 3′ termini of pre-mRNAs are properly cleaved but lack full-length poly(A) tails. These effects suggest that Pbp1p may act to repress the ability of Pab1p to negatively regulate polyadenylation.     

Translation initiation requires the PAB-dependent poly(A) ribonuclease in yeast.

Messenger RNA translation initiation and cytoplasmic poly(A) tail shortening require the poly(A)-binding protein (PAB) in yeast. The PAB-dependent poly(A) ribonuclease (PAN) has been purified to near homogeneity from S. cerevisiae based upon its PAB requirement, and its gene has been cloned. The essential PAN1 gene encodes a 161 kd protein organized into distinct domains containing repeated sequence elements. Deletion analysis of the gene revealed that only one-third of the protein is needed to maintain cell viability. Conditional mutations in PAN1 lead to an arrest of translation initiation and alterations in mRNA poly(A) tail lengths. These data suggest that PAN could mediate each of the PAB-dependent reactions within the cell, and they provide evidence for a direct relationship between translation initiation and mRNA metabolism.     

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.     

Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G.

Although the cap structure and the poly(A) tail are on opposite ends of the mRNA molecule, previous work has suggested that they interact to enhance translation and inhibit mRNA degradation. Here we present biochemical data that show that the proteins bound to the mRNA cap (eIF-4F) and poly(A) tail (Pab1p) are physically associated in extracts from the yeast Saccharomyces cerevisiae. Specifically, we find that Pab1p co-purifies and co-immunoprecipitates with the eIF-4G subunit of eIF-4F. The Pab1p binding site on the recombinant yeast eIF-4G protein Tif4632p was mapped to a 114-amino-acid region just proximal to its eIF-4E binding site. Pab1p only bound to this region when complexed to poly(A). These data support the model that the Pablp-poly(A) tail complex on mRNA can interact with the cap structure via eIF-4G.     

A specific role for the C-terminal region of the Poly(A)-binding protein in mRNA decay

mRNA poly(A) tails affect translation, mRNA export and mRNA stability, with translation initiation involving a direct interaction between eIF4G and the poly(A)-binding protein Pab1. The latter factor contains four RNA recognition motifs followed by a C-terminal region composed of a linker and a PABC domain. We show here that yeast mutants lacking the C-terminal domains of Pab1 display specific synthetic interactions with mutants in the 5′-3′ mRNA decay pathway. Moreover, these mutations impair mRNA decay in vivo without significantly affecting mRNA export or translation. Inhibition of mRNA decay occurs through slowed deadenylation. In vitro analyses demonstrate that removal of the Pab1 linker domain directly interferes with the ability of the Pop2–Ccr4 complex to deadenylate the Pab1-bound poly(A). Binding assays demonstrate that this results from a modulation of poly(A) packaging by the Pab1 linker region. Overall, our results demonstrate a direct involvement of Pab1 in mRNA decay and reveal the modular nature of this factor, with different domains affecting various cellular processes. These data suggest new models involving the modulation of poly(A) packaging by Pab1 to control mRNA decay.