Viral and cellular mRNA-specific activators harness PABP and eIF4G to promote translation initiation downstream of cap binding.

Regulation of mRNA translation is a major control point for gene expression and is critical for life. Of central importance is the complex between cap-bound eukaryotic initiation factor 4E (eIF4E), eIF4G, and poly(A) tail-binding protein (PABP) that circularizes mRNAs, promoting translation and stability. This complex is often targeted to regulate overall translation rates, and also by mRNA-specific translational repressors. However, the mechanisms of mRNA-specific translational activation by RNA-binding proteins remain poorly understood. Here, we address this deficit, focusing on a herpes simplex virus-1 protein, ICP27. We reveal a direct interaction with PABP that is sufficient to promote PABP recruitment and necessary for ICP27-mediated activation. PABP binds several translation factors but is primarily considered to activate translation initiation as part of the PABP-eIF4G-eIF4E complex that stimulates the initial cap-binding step. Importantly, we find that ICP27-PABP forms a complex with, and requires the activity of, eIF4G. Surprisingly, ICP27-PABP-eIF4G complexes act independently of the effects of PABP-eIF4G on cap binding to promote small ribosomal subunit recruitment. Moreover, we find that a cellular mRNA-specific regulator, Deleted in Azoospermia-like (Dazl), also employs the PABP-eIF4G interaction in a similar manner. We propose a mechanism whereby diverse RNA-binding proteins directly recruit PABP, in a non-poly(A) tail-dependent manner, to stimulate the small subunit recruitment step. This strategy may be particularly relevant to biological conditions associated with hypoadenylated mRNAs (e.g., germ cells/neurons) and/or limiting cytoplasmic PABP (e.g., viral infection, cell stress). This mechanism adds significant insight into our knowledge of mRNA-specific translational activation and the function of the PABP-eIF4G complex in translation initiation.

RNA immunoprecipitation identifies novel targets of DAZL in human foetal ovary.

Study question: Can novel meiotic RNA targets of DAZL (deleted in azoospermia-like) be identified in the human foetal ovary? Summary answer: SYCP1 (synaptonemal complex protein-1), TEX11 (testis expressed 11) and SMC1B (structural maintenance of chromosomes 1B) are novel DAZL targets in the human foetal ovary, thus DAZL may have previously unrecognised roles in the translational regulation of RNAs involved in chromosome cohesion and DNA recombination in the oocyte from the time of initiation of meiosis. What is known already: The phenotype of Dazl deficiency in mouse is infertility in both sexes and DAZL has also been linked to infertility in humans. Few studies have explored targets of this RNA-binding protein. The majority of these investigations have been carried out in mouse, and have focussed on the male thus the basis for its central function in regulating female fertility is largely unknown. Study design size, duration: We carried out RNA sequencing after immunoprecipitation of endogenous DAZL from human foetal ovarian tissue (17 weeks of gestation, obtained after elective termination of pregnancy) to identify novel DAZL targets involved in meiosis (n = 3 biological replicates). Participants/materials, setting, methods: Using quantitative RT-PCR, we examined the expression of selected RNAs identified by our immunoprecipitation across gestation, and visualised the expression of potential target SMC1B in relation to DAZL, with a combination of in situ hybridisation and immunohistochemistry. 3' untranslated region (3'UTR)-luciferase reporter assays and polysome profile analysis were used to investigate the regulation of three RNA targets by DAZL, representing key functionalities: SYCP1, TEX11 and SMC1B. Main results and the role of chance: We identified 764 potential RNA targets of DAZL in the human foetal ovary (false discovery rate 0.05 and log-fold change ≥ 2), with functions in synaptonemal complex formation (SYCP1, SYCP3), cohesin formation (SMC1B, RAD21), spindle assembly checkpoint (MAD2L1, TRIP13) and recombination and DNA repair (HORMAD1, TRIP13, TEX11, RAD18, RAD51). We demonstrated that the translation of novel targets SYCP1 (P = 0.004), TEX11 (P = 0.004) and SMC1B (P = 0.002) is stimulated by the presence of DAZL but not by a mutant DAZL with impaired RNA-binding activity. Large scale data: The raw data are available at GEO using the study ID: GSE81524. Limitations, reasons for caution: This analysis is based on identification of DAZL targets at the time when meiosis starts in the ovary: it may have other targets at other stages of oocyte development, and in the testis. Representative targets were validated, but detailed analysis was not performed on the majority of putative targets. Wider implications of the findings: These data indicate roles for DAZL in the regulation of several key functions in human oocytes. Through the translational regulation of novel RNA targets SMC1B and TEX11, DAZL may have a key role in regulating chromosome cohesion and DNA recombination; two processes fundamental in determining oocyte quality and whose establishment in foetal life may support lifelong fertility. Study funding and competing interest(s): This study was supported by the UK Medical Research Council (grant no G1100357 to R.A.A. and an intramural MRC programme grant to I.R.A.). The authors declare no competing interests.

Neutrophil-derived alpha defensins control inflammation by inhibiting macrophage mRNA translation.

Neutrophils are the first and most numerous cells to arrive at the site of an inflammatory insult and are among the first to die. We previously reported that alpha defensins, released from apoptotic human neutrophils, augmented the antimicrobial capacity of macrophages while also inhibiting the biosynthesis of proinflammatory cytokines. In vivo, alpha defensin administration protected mice from inflammation, induced by thioglychollate-induced peritonitis or following infection withSalmonella entericaserovar Typhimurium. We have now dissected the antiinflammatory mechanism of action of the most abundant neutrophil alpha defensin, Human Neutrophil Peptide 1 (HNP1). Herein we show that HNP1 enters macrophages and inhibits protein translation without inducing the unfolded-protein response or affecting mRNA stability. In a cell-free in vitro translation system, HNP1 powerfully inhibited both cap-dependent and cap-independent mRNA translation while maintaining mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell type (neutrophils) directly regulating mRNA translation in another (macrophages). By preventing protein translation, HNP1 functions as a "molecular brake" on macrophage-driven inflammation, ensuring both pathogen clearance and the resolution of inflammation with minimal bystander tissue damage.

Poly(A)-binding proteins and mRNA localization: who rules the roost?

RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals.

Poly(A)-binding proteins are required for diverse biological processes in metazoans.

PABPs [poly(A)-binding proteins] bind to the poly(A) tail of eukaryotic mRNAs and are conserved in species ranging from yeast to human. The prototypical cytoplasmic member, PABP1, is a multifunctional RNA-binding protein with roles in global and mRNA-specific translation and stability, consistent with a function as a central regulator of mRNA fate in the cytoplasm. More limited insight into the molecular functions of other family members is available. However, the consequences of disrupting PABP function in whole organisms is less clear, particularly in vertebrates, and even more so in mammals. In the present review, we discuss current and emerging knowledge with respect to the functions of PABP family members in whole animal studies which, although incomplete, already underlines their biological importance and highlights the need for further intensive research in this area.

A molecular doorstop ensures a trickle through translational repression.

Switching mRNA translation off and on is central to regulated gene expression, but what mechanisms moderate the extent of switch-off? Yao et al. describe how basal expression from interferon-gamma-induced transcripts is maintained during mRNA-specific translational repression. This antagonistic mechanism utilizes a truncated RNA-binding factor generated by a unique alternative polyadenylation event.

DAZAP1, an RNA-binding protein required for development and spermatogenesis, can regulate mRNA translation.

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 that Xenopus laevis and 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.

Poly(A)-binding protein (PABP): a common viral target.

Cytoplasmic PABP [poly(A)-binding protein] is a multifunctional protein with well-studied roles in mRNA translation and stability. In the present review, we examine recent evidence that the activity of PABP is altered during infection with a wide range of viruses, bringing about changes in its stability, complex formation and intracellular localization. Targeting of PABP by both RNA and DNA viruses highlights the role of PABP as a central regulator of gene expression.

Regulation of translation initiation by herpesviruses.

Viruses are dependent upon the host cell protein synthesis machinery, thus they have developed a range of strategies to manipulate host translation to favour viral protein synthesis. Consequently, the study of viral translation has been a powerful tool for illuminating many aspects of cellular translational control. Although much work to date has focused on translational regulation by RNA viruses, DNA viruses have also evolved complex mechanisms to regulate protein synthesis. Here we summarize work on a large family of DNA viruses, the Herpesviridae, which have evolved mechanisms to sustain efficient cap-dependent translation and to regulate the translation of specific viral mRNAs.

Direct stimulation of translation by the multifunctional herpesvirus ICP27 protein.

Herpes simplex virus type 1 (HSV-1) ICP27 protein is an essential regulator of viral gene expression with roles at various levels of RNA metabolism in the nucleus. Using the tethered function assay, we showed a cytoplasmic activity for ICP27 in directly enhancing mRNA translation in vivo in the absence of other viral factors. The region of ICP27 required for translational stimulation maps to the C terminus. Furthermore, in infected cells, ICP27 is associated with polyribosomes, indicating a function in translation during the lytic cycle.

Coordinated regulation of replication protein A activities by its subunits p14 and p32.

The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPADeltap14, consisting of p70 and p32. RPADeltap14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPADeltap14 complex p32 only minimally recognized the 3'-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPADeltap14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase alpha-primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on single-stranded DNA requires all three subunits to support DNA replication.

Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase alpha-primase.

Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase alpha-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase alpha-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex.

Species specificity of simian virus 40 DNA replication in vitro requires multiple functions of human DNA polymerase alpha.

Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase alpha-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase alpha-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase alpha-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase alpha depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase alpha-primase but rather on sequences in the C-terminal region of human p180.