It has previously been shown that Pab1p from yeast can interact with the 5′ cap-binding complex eIF4F, which contains eIF4G (23
). Here, we extend this finding by showing that this function requires RRM2 of Pab1p. The functional consequences of the interaction between RRM2 and eIF4G are demonstrated by the need for RRM2 in poly(A) tail-dependent translation in yeast extracts. Our analysis of the Pab1-16p mutation also reveals that while association of Pab1p with eIF4G is a prerequisite for translational stimulation, other features of this interaction may also be required.
The most obvious finding of this study is the requirement for RRM2 of Pab1p in mediating binding to eIF4G and in stimulating poly(A) tail-dependent translation in vitro. What is less clear is the role of the other domains of Pab1p, which include three RRMs and a 17-kDa C-terminal region. Our in vitro translation data could suggest that RRM1 and RRM4 and the C-terminal region of Pab1p are also involved in mediating the Pab1p-poly(A) tail translation function. Specifically, we found that deletion of RRM4 had an effect in the reconstitution assay, while deletion of RRM1 had a significant effect in extracts prepared from strains containing this Pab1p mutant (compare Fig. A and B). Yeast RRM4 has been previously shown to have significant RNA-binding activity (5
), and this activity may contribute to the translation function of the protein. As a result of its proximity to RRM2, RRM1 may provide structural support to RRM2 and thereby enhance the function of this latter domain. The C-terminal region of Xenopus
Pab1p has been suggested to mediate Pab1p dimerization (12
), and this could also serve to heighten the translation function of Pab1p. Further experiments will be needed in order to understand how other regions of Pab1p serve to enhance the translational function of RRM2.
We are unable to conclude from our data that RRM2 directly contacts eIF4G because of our inability to demonstrate an interaction between the isolated RRM2 and eIF4G. One possible explanation for this observation is that an isolated RRM2 lacks structural integrity. Consistent with this proposal, we have been unable to observe significant poly(A) binding by this domain in isolation (data not shown), which presumably prevents its RNA-dependent interaction with eIF4G. The minimal Pab1p which we could show has both eIF4G-binding activity and translational activity (Pab1-105p) contains both RRM1 and RRM2. Thus, it is possible that additional essential stabilizing contacts with RRM2 are made through RRM1. Because RRM1 can be singly deleted from Pab1p without destroying the interaction with eIF4G and Pab1p-dependent translation, we assume that these proposed stabilizing contacts can also be supplied by the other Pab1p RRMs.
Why does Pab1p require poly(A) in order for it to interact with eIF4G? RNA binding may place Pab1p in an appropriate conformation to bind to eIF4G. Alternatively, Pab1p may place the poly(A) into a conformation suitable for contacting a latent eIF4G RNA-binding site. The eIF4G protein may also contact both Pab1p and poly(A). The requirement for a Pab1p-poly(A) complex for the interaction with eIF4G may exist so as to prevent association of either non-mRNA-associated Pab1p or naked poly(A) with eIF4G. This would create a more stringent and specific requirement for Pab1p-dependent activation of translation.
An exciting yet unexpected result was provided by the analysis of Pab1-16p. This protein was originally constructed with the goal of disrupting its RNA-binding activity (5
), but our investigation has now suggested an additional role for its altered residues in Pab1p-dependent translation. This protein exhibits a reduction in the affinity for poly(A) RNA (5
) yet still interacts with eIF4G (Fig. A). However, this protein is incapable of stimulating the translation of uncapped, polyadenylated (LUCpA) mRNA in vitro (Fig. B and C). This result indicates that a simple binding event is insufficient for mediating poly(A) tail-dependent translation. The nature of the defect of Pab1-16p will require further investigation. As mentioned above, the possibility that Pab1-16p has a decreased affinity for eIF4G will be pursued. The deficiency is unlikely to be due to the reduction in affinity for poly(A), as Pab1-105p has a similar affinity yet still activates translation in vitro. This finding is also interesting in light of genetic analysis of pab1-16
. This allele exhibits synthetic lethality with cdc33-1
), which is a mutant allele of the gene encoding eIF4E. In the latter mutant, cap-dependent translation is compromised. Thus, a possible reason for the observed synthetic lethality is the deleterious effects of losing or reducing both cap-dependent and poly(A) tail-dependent translation in vivo.
Future analysis of the interaction between Pab1p and eIF4G will involve site-directed mutagenesis of Pab1p. This approach will allow for the identification of the amino acid side chains that contact eIF4G, thus more precisely defining the interaction surface. It should also identify other residues within Pab1p that are dispensable for association with eIF4G but are required for translational stimulation. Site-directed mutagenesis, which has been used to examine the RNA binding of Pab1p (5
), should alleviate any potential structural perturbations caused by the deletion of the ~90-amino-acid RRMs and thus create fewer ambiguities in the data interpretation. Nevertheless, the deletion analysis of Pab1p presented here has given us the ability to define a region of the protein that is involved in translation initiation in vitro and has therefore provided us with the region of the protein which will be subject to more intensive investigation.