Role In mRNA Translation Research Articles

Identification of a C-terminal Poly(A)-binding Protein (PABP)-PABP Interaction Domain: ROLE IN COOPERATIVE BINDING TO POLY(A) AND EFFICIENT CAP DISTAL TRANSLATIONAL REPRESSION

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.

37 oral The role of MRNA translation in hypoxia tolerance

37 oral The role of MRNA translation in hypoxia tolerance

The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1.

Previously, we have shown that the vimentin 3' untranslated region (3'UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA. This region was shown to specifically bind protein(s) by band shift analyses. UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass. Here, we have used this sequence as 'bait' to isolate RNA binding proteins using the yeast three-hybrid method. This technique relies on a functional assay detecting bona fide RNA-protein interaction in vivo. Three cDNA isolates, HAX-1, eEF-1gamma and hRIP, code for proteins of a size consistent with in vitro cross- linking studies. In all cases, recombinant proteins were capable of binding RNA in vitro. Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-1gamma and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin's 3'UTR. Both in vivo synthesized eEF-1gamma and HAX-1 proteins were 'pulled out' of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin's 3'UTR. Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1gamma or HAX-1. Thus, in addition to their known functions, both eEF-1gamma and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.

Does phosphorylation of the cap-binding protein eIF4E play a role in translation initiation?

Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5′‐cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E can interact either with the scaffold protein eIF4G or with repressor proteins termed eIF4E‐binding proteins (4E‐BPs). High levels of expression can disrupt cellular growth control and are associated with human cancers. A fraction of the cellular eIF4E is found in the nucleus where it may play a role in the transport of certain mRNAs to the cytoplasm. eIF4E undergoes regulated phosphorylation (at Ser209) by members of the Mnk group of kinases, which are activated by multiple MAP kinases (hence Mnk = MAP‐kinase signal integrating kinase). The functional significance of its phosphorylation has been the subject of considerable interest. Recent genetic studies in Drosophila point to a key role for phosphorylation of eIF4E in growth and viability. Initial structural data suggested that phosphorylation of Ser209 might allow formation of a salt bridge with a basic residue (Lys159) that would clamp eIF4E onto the mRNA and increase its affinity for ligand. However, more recent structural data place Ser209 too far away from Lys159 to form such an interaction, and biophysical studies indicate that phosphorylation actually decreases the affinity of eIF4E for cap or capped RNA. The implications of these studies are discussed in the light of other, in vitro and in vivo, investigations designed to address the role of eIF4E phosphorylation in mRNA translation or its control.

Rapid induction of apoptosis mediated by peptides that bind initiation factor eIF4E.

Overexpression of the translation initiation factor eIF4E leads to cell transformation and occurs in a number of human cancers [1]. mRNA translation and cell growth can be regulated through the availability of eIF4E to form initiation complexes by binding to eIF4G. The availability of eIF4E is blocked through the binding of members of a family of eIF4E-binding proteins (4E-BPs) [2] [3]. Indeed, cell transformation caused by the overexpression of eIF4E can be reversed by the overexpression of 4E-BPs [4] [5] [6] [7] [8]. To study the role of eIF4E in cell transformation, we developed a series of peptides based on the conserved eIF4E-binding motifs in 4E-BPs and eIF4G [9] linked to the penetratin peptide-carrier sequence, which mediates the rapid transport of peptides across cell membranes. Surprisingly, introduction of these eIF4E-binding peptides into MRC5 cells led to rapid, dose-dependent cell death, with characteristics of apoptosis. Single alanine substitutions at key positions in the peptides impair their binding to eIF4E and markedly reduce their ability to induce apoptosis. A triple alanine substitution, which abolishes binding to eIF4E, renders the peptide unable to induce apoptosis. Our data provide strong evidence that the peptides induce apoptosis through binding to eIF4E. They do not induce apoptosis through inhibition of protein synthesis, as chemical inhibitors of translation did not induce apoptosis or affect peptide-induced cell death. Thus these new data indicate that eIF4E has a direct role in controlling cell survival that is not linked to its known role in mRNA translation.

A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation.

Dimorphic growth of the budding yeast Saccharomyces cerevisiae is regulated by the quality of the nitrogen supply. On a preferred nitrogen source diploid cells grow as ellipsoidal cells by using a bipolar pattern of budding, whereas on a poor nitrogen source a unipolar pattern of budding is adopted, resulting in extended pseudohyphal chains of filamentous cells. Here we report that the quality of the nitrogen source is signaled by the glutamine tRNA isoform with a 5'-CUG anticodon (tRNACUG). Mutations that alter this tRNA impair assessment of the nitrogen supply without measurably affecting protein synthesis, so that mutant cells display pseudohyphal growth even on a preferred nitrogen source. The nitrogen status for other nitrogen-responsive processes such as catabolic gene expression and sporulation also is signaled by this tRNA: mutant cells inappropriately induce the nitrogen-repressed gene CAR1 and undergo precocious sporulation in nitrogen-rich media. Therefore, in addition to its role in mRNA translation, this tRNA also transduces nitrogen signals that regulate development.

Polyadenylation of mRNA in prokaryotes.

The 3'-ends of both prokaryotic and eukaryotic mRNA are polyadenylated, but the poly(A) tracts of prokaryotic mRNA are generally shorter, ranging from 15 to 60 adenylate residues and associated with only 2-60% of the molecules of a given mRNA species. The sites of polyadenylation of bacterial mRNA are diverse and include the 3'-ends of primary transcripts, the sites of endonucleolytic processing in the 3' untranslated and intercistronic regions, and sites within the coding regions of mRNA degradation products. The diversity of polyadenylation sites suggests that mRNA polyadenylation in prokaryotes is a relatively indiscriminate process that can occur at all mRNA's 3'-ends and does not require specific consensus sequences as in eukaryotes. Two poly(A) polymerases have been identified in Escherichia coli. They are encoded by unlinked genes, neither of which is essential for growth, suggesting significant functional overlap. Polyadenylation promotes the degradation of a regulatory RNA that inhibits the replication of bacterial plasmids and may play a similar role in the degradation of mRNA. However, under certain conditions, poly(A) tracts may lead to mRNA stabilization. Their ability to bind S1 ribosomal protein suggests that poly(A) tracts may also play a role in mRNA translation.

Molecular Characterization of PAB2, a Member of the Multigene Family Coding for Poly(A)-Binding Proteins in Arabidopsis thaliana

The poly(A) tail of eukaryotic mRNAs associates with poly(A)-binding (PAB) proteins whose role in mRNA translation and stability is being intensively investigated. Very little is known about the structure and function of the PAB genes in plants. We have cloned multiple PAB-related sequences from Arabidopsis thaliana. Results suggest that PAB proteins are encoded by a multigene family. One member of this family (PAB2) is expressed in root and shoot tissues. The complete nucleotide sequence of PAB2 was determined. Study of the predicted PAB2 protein reveals a similarity in structure among vertebrate, insect, yeast, and plant PAB proteins. All contain two highly conserved domains: an amino-terminal sequence formed by four RNA recognition motifs and an uncharacterized carboxyl-terminal region of 69 to 71 amino acids. Possible roles for the carboxyl-terminal conserved domain are discussed in view of recently published data concerning the structure and function of PAB proteins.

Role of the 3'-poly(A) sequence in translational regulation of mRNAs in Xenopus laevis oocytes.

We investigated the effect of 3'-noncoding and poly(A) sequences on the translation and stability of membrane-bound mRNA (maize zein) and free mRNA (Xenopus beta-globin) by injecting SP6 transcripts into stage 6 Xenopus oocytes. With zein mRNA, the presence or absence of a 3'-noncoding or poly(A) sequence had little effect on mRNA stability over 24 h, and the 3'-noncoding sequence played essentially no role in mRNA translation. With short periods of incubation, e.g. 1-2 h, the presence or absence of a poly(A) tail also had little effect on zein mRNA translation; but after longer periods, translation of the poly(A-) mRNA was significantly reduced. A similar pattern of mRNA translation was observed for poly(A+) and poly(A-) Xenopus beta-globin mRNAs. These differences in translational efficiency correlated with the formation of maximally loaded polysomes (seven-eight ribosomes/mRNA) for the poly(A+) zein mRNA and a failure to form large polysomes with the poly(A-) zein mRNA. These results are consistent with a model in which the 3'-poly(A) sequence of mRNAs facilitates reinitiation of ribosomes during protein synthesis.