Alan B. Sachs

Starting at the Beginning, Middle, and End: Translation Initiation in Eukaryotes

The exclusive role of the mRNA cap structure in the 40S subunit recruitment process during translation initiation is no longer tenable. The ability of IRES elements and poly(A) tails in yeast to stimulate 40S subunit binding forces a change in this viewpoint. While it is almost certain that the vast majority of mRNAs are translated by 40S subunits scanning from the cap structure, it now seems likely that the 40S subunit can also be brought to the mRNA via an interaction with the mRNA poly(A) tail. Subsequently or simultaneously with this interaction, the cap structure, due to its high affinity for eIF4E, could act as a docking site for the recruited subunit (Figure 3Figure 3). In this model, both the cap structure and the poly(A) tail share the function of 40S subunit recruitment, while the cap structure has the exclusive role of docking the subunit onto a unique position in the mRNA. We note that the role of poly(A) tails in translation in higher eukaryotes is assumed but not yet shown since only the role of poly(A) tails in yeast translation has been thoroughly studied.Cellular IRES elements could replace the role of the cap structure on those mRNAs where the cap is either masked or eIF4E is inactive (Figure 3Figure 3). Recruitment of the 40S subunit to the mRNA by the poly(A) tail could occur prior to or simultaneously with the placement of the subunit at a position on the mRNA determined by the location of the IRES element. In this model, the cap structure and the IRES element have identical functions in the translation process: they assist in recruitment of the 40S subunit to the mRNA, and they provide a loading site for 40S subunits at a unique position on the mRNA.Future work in this exciting area of translation research should help to test the basic tenets of this model. Since many of the central experiments so far have been carried out in cell-free systems and commonly under conditions where mRNA is limiting, it will be important to determine the interplay of the diferent modes of ribosome recruitment under conditions of mRNA competition for initiation factors. Likewise, we do not yet understand much regarding possible differences between the first and subsequent rounds of initiation, and the roles of the cap, IRES and poly(A) tail in ribosome recycling. Along the way, it is anticipated that new insights into how an mRNAs expression can be controlled in the cytoplasm of eukaryotic cells will be uncovered. With more information, it will hopefully become clearer how mRNA sequences, including the 5′ NCR and the 3′ UTR, can regulate an mRNAs expression.

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.

The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation

Depletion of the essential poly(A) binding protein (PAB) in S. cerevisiae by promoter inactivation or by the utilization of a temperature-sensitive mutation (pab1-F364L) results in the inhibition of translation initiation and poly(A) tail shortening. Reversion analysis of pab1-F364L yielded seven independent, extragenic cold-sensitive mutations (spb1-spb7) that also suppress a PAB1 deletion. These mutations allow translation initiation without significantly changing poly(A) tail lengths in the absence of PAB, and they affect the amount of 60S ribosomal subunit. Consistent with this, SPB2 encodes the ribosomal protein L46. These data suggest that the 60S subunit mediates the PAB requirement of translation initiation, thereby ensuring that only intact poly(A)+ mRNA will be translated efficiently in vivo.

A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

A single gene from yeast for both nuclear and cytoplasmic polyadenylate-binding proteins: domain structure and expression.

Nuclear and cytoplasmic poly(A)-binding proteins have been purified from Saccharomyces cerevisiae, and antisera have been used to isolate a gene that encodes them. The gene occurs in a single copy on chromosome 5 and gives rise to a unique, unspliced 2.1 kb transcript. The nuclear protein appears to be derived from the cytoplasmic one by proteolytic cleavage into 53 and 17 kd polypeptides that remain associated during isolation. DNA sequence determination reveals four tandemly arrayed 90 amino acid regions of homology that probably represent poly(A)-binding domains. A 55 residue A-rich region upstream of the initiator methionine codon in the mRNA shows an affinity for poly(A)-binding protein comparable to that of poly(A)180-220, raising the possibility of feedback regulation of translation.

Ribosome loading onto the mRNA cap is driven by conformational coupling between eIF4G and eIF4E.

The eukaryotic initiation factor 4G (eIF4G) is the core of a multicomponent switch controlling gene expression at the level of translation initiation. It interacts with the small ribosomal subunit interacting protein, eIF3, and the eIF4E/cap-mRNA complex in order to load the ribosome onto mRNA during cap-dependent translation. We describe the solution structure of the complex between yeast eIF4E/cap and eIF4G (393-490). Binding triggers a coupled folding transition of eIF4G (393-490) and the eIF4E N terminus resulting in a molecular bracelet whereby eIF4G (393-490) forms a right-handed helical ring that wraps around the N terminus of eIF4E. Cofolding allosterically enhances association of eIF4E with the cap and is required for maintenance of optimal growth and polysome distributions in vivo. Our data explain how mRNA, eIF4E, and eIF4G exists as a stable mRNP that may facilitate multiple rounds of ribosomal loading during translation initiation, a key determinant in the overall rate of protein synthesis.

Poly(A) Tail Length Control in Saccharomyces cerevisiae Occurs by Message-Specific Deadenylation

ABSTRACT We report that newly synthesized mRNA poly(A) tails are matured to precise lengths by the Pab1p-dependent poly(A) nuclease (PAN) ofSaccharomyces cerevisiae. These results provide evidence for an initial phase of mRNA deadenylation that is required for poly(A) tail length control. In RNA 3′-end processing extracts lacking PAN, transcripts are polyadenylated to lengths exceeding 200 nucleotides. By contrast, in extracts containing PAN, transcripts were produced with the expected wild-type poly(A) tail lengths of 60 to 80 nucleotides. The role for PAN in poly(A) tail length control in vivo was confirmed by the finding that mRNAs are produced with longer poly(A) tails in PAN-deficient yeast strains. Interestingly, wild-type yeast strains were found to produce transcripts which varied in their maximal poly(A) tail length, and this message-specific length control was lost in PAN-deficient strains. Our data support a model whereby mRNAs are polyadenylated by the 3′-end processing machinery with a long tail, possibly of default length, and then in a PAN-dependent manner, the poly(A) tails are rapidly matured to a message-specific length. The ability to control the length of the poly(A) tail for newly expressed mRNAs has the potential to be an important posttranscriptional regulatory step in gene expression.

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

ABSTRACT 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.

Capped mRNA Degradation Intermediates Accumulate in the Yeast spb8-2 Mutant

ABSTRACT mRNA in the yeast Saccharomyces cerevisiae is primarily degraded through a pathway that is stimulated by removal of the mRNA cap structure. Here we report that a mutation in the SPB8(YJL124c) gene, initially identified as a suppressor mutation of a poly(A)-binding protein (PAB1) gene deletion, stabilizes the mRNA cap structure. Specifically, we find that thespb8-2 mutation results in the accumulation of capped, poly(A)-deficient mRNAs. The presence of this mutation also allows for the detection of mRNA species trimmed from the 3′ end. These data show that this Sm-like protein family member is involved in the process of mRNA decapping, and they provide an example of 3′-5′ mRNA degradation intermediates in yeast.