Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis

RNA-binding proteins that bind to the 3′ untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis. © 1995 Wiley-Liss, Inc.     

Autoregulation of poly(A)-binding protein synthesis in vitro.

The poly(A)-binding protein (PABP), in a complex with the 3'poly(A) tail of eukaryotic mRNAs, plays important roles in the control of translation and message stability. All known examples of PABP mRNAs contain an extensive A-rich sequence in their 5' untranslated regions. Studies in mammalian cells undergoing growth stimulation or terminal differentiation indicate that PABP expression is regulated at the translational level. Here we examine the hypothesis that synthesis of the PABP is autogenously controlled. We show that the endogenous inactive PABP mRNA in rabbit reticulocytes can be specifically stimulated by addition of low concentrations of poly(A) and that this stimulation is also observed with in vitro transcribed human PABP mRNA. By deleting the A-rich region from the leader of human PABP mRNA and adding it upstream of the initiator AUG in a reporter mRNA we show that the adenylate tract is sufficient and necessary for mRNA repression and poly(A)-mediated activation in the reticulocyte cell-free system. UV cross-linking experiments demonstrate that the leader adenylate tract binds PABP. Furthermore, addition of recombinant GST-PABP to the cell-free system represses translation of mRNAs containing the A-rich sequence in their 5'UTR, but has no effect on control mRNA. We thus conclude that in vitro PABP binding to the A-rich sequence in the 5' UTR of PABP mRNA represses its own synthesis.     

HIV- 1 Protease Inhibits Cap- and Poly(A)-Dependent Translation upon eIF4GI and PABP Cleavage

A number of viral proteases are able to cleave translation initiation factors leading to the inhibition of cellular translation. This is the case of human immunodeficiency virus type 1 protease (HIV-1 PR), which hydrolyzes eIF4GI and poly(A)-binding protein (PABP). Here, the effect of HIV-1 PR on cellular and viral protein synthesis has been examined using cell-free systems. HIV-1 PR strongly hampers translation of pre-existing capped luc mRNAs, particularly when these mRNAs contain a poly(A) tail. In fact, HIV-1 PR efficiently blocks cap- and poly(A)-dependent translation initiation in HeLa extracts. Addition of exogenous PABP to HIV-1 PR treated extracts partially restores the translation of polyadenylated luc mRNAs, suggesting that PABP cleavage is directly involved in the inhibition of poly(A)-dependent translation. In contrast to these data, PABP cleavage induced by HIV-1 PR has little impact on the translation of polyadenylated encephalomyocarditis virus internal ribosome entry site (IRES)-containing mRNAs. In this case, the loss of poly(A)-dependent translation is compensated by the IRES transactivation provided by eIF4G cleavage. Finally, translation of capped and polyadenylated HIV-1 genomic mRNA takes place in HeLa extracts when eIF4GI and PABP have been cleaved by HIV-1 PR. Together these results suggest that proteolytic cleavage of eIF4GI and PABP by HIV-1 PR blocks cap- and poly(A)-dependent initiation of translation, leading to the inhibition of cellular protein synthesis. However, HIV-1 genomic mRNA can be translated under these conditions, giving rise to the production of Gag polyprotein.     

Recognition of polyadenylate RNA by the poly(A)-binding protein.

The cocrystal structure of human poly(A)-binding protein (PABP) has been determined at 2.6 A resolution. PABP recognizes the 3' mRNA poly(A) tail and plays critical roles in eukaryotic translation initiation and mRNA stabilization/degradation. The minimal PABP used in this study consists of the N-terminal two RRM-type RNA-binding domains connected by a short linker (RRM1/2). These two RRMs form a continuous RNA-binding trough, lined by an antiparallel beta sheet backed by four alpha helices. The polyadenylate RNA adopts an extended conformation running the length of the molecular trough. Adenine recognition is primarily mediated by contacts with conserved residues found in the RNP motifs of the two RRMs. The convex dorsum of RRM1/2 displays a phylogenetically conserved hydrophobic/acidic portion, which may interact with translation initiation factors and regulatory proteins.     

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.     

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.     

Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2

The poly(A)-binding protein (PABP) is a unique translation initiation factor in that it binds to the mRNA 3' poly(A) tail and stimulates recruitment of the ribosome to the mRNA at the 5' end. PABP activity is tightly controlled by the PABP-interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA. Here, we describe a close interplay between PABP and Paip2 protein levels in the cell. We demonstrate a mechanism for this co-regulation that involves an E3 ubiquitin ligase, EDD, which targets Paip2 for degradation. PABP depletion by RNA interference (RNAi) causes co-depletion of Paip2 protein without affecting Paip2 mRNA levels. Upon PABP knockdown, Paip2 interacts with EDD, which leads to Paip2 ubiquitination. Supporting a critical role for EDD in Paip2 degradation, knockdown of EDD expression by siRNA leads to an increase in Paip2 protein stability. Thus, we demonstrate that the turnover of Paip2 in the cell is mediated by EDD and is regulated by PABP. This mechanism serves as a homeostatic feedback to control the activity of PABP in cells.