Poly(A)-binding protein 1 (PABP1) has a fundamental role in the regulation of mRNA translation and stability, both of which are crucial for a wide variety of cellular processes. Although generally a diffuse cytoplasmic protein, it can be found in discrete foci such as stress and neuronal granules. Mammals encode several additional cytoplasmic PABPs that remain poorly characterised, and with the exception of PABP4, appear to be restricted in their expression to a small number of cell types. We have found that PABP4, similarly to PABP1, is a diffusely cytoplasmic protein that can be localised to stress granules. However, UV exposure unexpectedly relocalised both proteins to the nucleus. Nuclear relocalisation of PABPs was accompanied by a reduction in protein synthesis but was not linked to apoptosis. In examining the mechanism of PABP relocalisation, we found that it was related to a change in the distribution of poly(A) RNA within cells. Further investigation revealed that this change in RNA distribution was not affected by PABP knockdown but that perturbations that block mRNA export recapitulate PABP relocalisation. Our results support a model in which nuclear export of PABPs is dependent on ongoing mRNA export, and that a block in this process following UV exposure leads to accumulation of cytoplasmic PABPs in the nucleus. These data also provide mechanistic insight into reports that transcriptional inhibitors and expression of certain viral proteins cause relocation of PABP to the nucleus.

Translational control of many mRNAs in developing metazoan embryos is achieved by alterations in their poly(A) tail length. A family of cytoplasmic poly(A)-binding proteins (PABPs) bind the poly(A) tail and can regulate mRNA translation and stability. However, despite the extensive biochemical characterization of one family member (PABP1), surprisingly little is known about their in vivo roles or functional relatedness. Because no information is available in vertebrates, we address their biological roles, establishing that each of the cytoplasmic PABPs conserved in Xenopus laevis [PABP1, embryonic PABP (ePABP), and PABP4] is essential for normal development. Morpholino-mediated knockdown of PABP1 or ePABP causes both anterior and posterior phenotypes and embryonic lethality. In contrast, depletion of PABP4 results mainly in anterior defects and lethality at later stages. Unexpectedly, cross-rescue experiments reveal that neither ePABP nor PABP4 can fully rescue PABP1 depletion, establishing that PABPs have distinct functions. Comparative analysis of the uncharacterized PABP4 with PABP1 and ePABP shows that it shares a mechanistically conserved core role in promoting global translation. Consistent with this analysis, each morphant displays protein synthesis defects, suggesting that their roles in mRNA-specific translational regulation and/or mRNA decay, rather than global translation, underlie the functional differences between PABPs. Domain-swap experiments reveal that the basis of the functional specificity is complex, involving multiple domains of PABPs, and is conferred, at least in part, by protein–protein interactions.

The gonadotropin receptors, luteinising hormone receptor, and follicle-stimulating hormone receptor, play a central role in governing reproductive competency/fertility. Gonadotropin hormone analogues have been utilised clinically for decades, in assisted reproductive therapies, and in the treatment of various infertility disorders. While these treatments are effective, the clinical protocols demand multiple injections, and the hormone preparations can lack uniformity and stability. The past two decades have seen a drive to develop chimeric and modified peptide analogues with more desirable pharmacokinetic profiles with some displaying clinical efficacy, such as corifollitropin alfa which is now in clinical use. More recently, low molecular weight orally-active molecules with activity at gonadotropin receptors have been developed. Some have excellent characteristics in animals and in human studies but have not reached the market: largely due to acquisitions by large pharma. Nonetheless such molecules have the potential to mitigate risks currently associated with gonadotropin-based fertility treatments such as ovarian hyperstimulation syndrome and the demands of injection-based therapies. There is also scope for novel utilisation beyond the current remit of gonadotropin analogues in fertility treatments, including application as novel contraceptives, and in the treatment of polycystic ovary syndrome, the restoration of function to inactivating mutations of gonadotropin receptors, ovarian and prostate cancers, and in bone loss and weight gain in postmenopausal women. Here, we review the properties and clinical application of current gonadotropin preparations and their analogues, as well as the development of novel orally-active small-molecule non-peptide analogues.

DAZ-associated protein 1 (DAZAP1) is an RNA-binding protein required for normal growth, development, and fertility in mice. However, its molecular functions have not been elucidated. Here we find thatXenopus laevisand human DAZAP1, which are each expressed as short and long forms, act as mRNA-specific activators of translation in a manner that is sensitive to the number of binding sites present within the 3′ UTR. Domain mapping suggests that this conserved function is mainly associated with C-terminal regions of DAZAP1. Interestingly, we find that the expression of xDAZAP1 and its polysome association are developmentally controlled, the latter suggesting that the translational activator function of DAZAP1 is regulated. However, ERK phosphorylation of DAZAP1, which can alter protein interactions with its C terminus, does not play a role in regulating its ability to participate in translational complexes. Since relatively few mRNA-specific activators have been identified, we explored the mechanism by which DAZAP1 activates translation. By utilizing reporter mRNAs with internal ribosome entry sites, we establish that DAZAP1 stimulates translation initiation. Importantly, this activity is not dependent on the recognition of the 5′ cap by initiation factors, showing that it functions downstream from this frequently regulated event, but is modulated by changes in the adenylation status of mRNAs. This suggests a function in the formation of “end-to-end” complexes, which are important for efficient initiation, which we show to be independent of a direct interaction with the bridging protein eIF4G.

Mutations in G protein–coupled receptors (GPCRs) have been identified for many endocrine hormone signaling deficiencies. Inactivating mutations can impair ligand binding, receptor activation/coupling to signaling pathways, or can cause receptor misfolding and consequent impaired expression at the cell membrane. Here we examine the cell surface expression, ligand binding, and signaling of a range of mutant human luteinizing hormone receptors (LHRs) identified as causing reproductive dysfunction in human patients. The data obtained reveal how mutations in GPCRs can have diverse and severely deleterious effects on receptor function. Furthermore, it was found that impaired functionality of the majority of the mutant LHRs was due to reduced expression at the cell surface (14/20) while only two mutations caused impaired binding affinity and two impaired in signaling. An additional two mutations were found to cause no impairment of receptor function. These data demonstrate that the majority of LHR mutations lead to intracellular retention and highlight the potential for novel pharmacological chaperone therapeutics that can “rescue” expression/function of retained mutant GPCRs.

Significance: The majority of genes are controlled at the level of mRNA translation, with accurate regulation being critical to cellular function and health. The repression or activation of subsets of mRNAs, so-called “mRNA-specific” regulation, is often mediated by RNA-binding proteins. However, the mechanisms underlying mRNA-specific activation have only been determined in a very few cases. Here, we uncover a mechanism of mRNA-specific activation used by viral and cellular proteins, which share no sequence similarity, suggesting that it may represent a widespread mechanism. Importantly, in so doing, we also expand our knowledge of how two key translation factors, poly(A)-binding protein (PABP) and eukaryotic initiation factor 4G (eIF4G), function during translation initiation, showing they have pleiotropic effects on small ribosomal subunit recruitment.