Nadeem Siddiqui

Structural basis for the recruitment of the human CCR4-NOT deadenylase complex by tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein that controls the inflammatory response by limiting the expression of several proinflammatory cytokines. TTP post-transcriptionally represses gene expression by interacting with AU-rich elements (AREs) in 3′ untranslated regions of target mRNAs and subsequently engenders their deadenylation and decay. TTP accomplishes these tasks, at least in part, by recruiting the multisubunit CCR4–NOT deadenylase complex to the mRNA. Here we identify an evolutionarily conserved C-terminal motif in human TTP that directly binds a central domain of CNOT1, a core subunit of the CCR4–NOT complex. A high-resolution crystal structure of the TTP–CNOT1 complex was determined, providing the first structural insight, to our knowledge, into an ARE-binding protein bound to the CCR4–NOT complex. Mutations at the CNOT1-TTP interface impair TTP-mediated deadenylation, demonstrating the significance of this interaction in TTP-mediated gene silencing.

eIF4E/4E-BP Ratio Predicts the Efficacy of mTOR Targeted Therapies

Active-site mTOR inhibitors (asTORi) hold great promise for targeting dysregulated mTOR signaling in cancer. Because of the multifaceted nature of mTORC1 signaling, identification of reliable biomarkers for the sensitivity of tumors to asTORi is imperative for their clinical implementation. Here, we show that cancer cells acquire resistance to asTORi by downregulating eukaryotic translation initiation factor (eIF4E)-binding proteins (4E-BPs-EIF4EBP1, EIF4EBP2). Loss of 4E-BPs or overexpression of eIF4E renders neoplastic growth and translation of tumor-promoting mRNAs refractory to mTOR inhibition. Conversely, moderate depletion of eIF4E augments the anti-neoplastic effects of asTORi. The anti-proliferative effect of asTORi in vitro and in vivo is therefore significantly influenced by perturbations in eIF4E/4E-BP stoichiometry, whereby an increase in the eIF4E/4E-BP ratio dramatically limits the sensitivity of cancer cells to asTORi. We propose that the eIF4E/4E-BP ratio, rather than their individual protein levels or solely their phosphorylation status, should be considered as a paramount predictive marker for forecasting the clinical therapeutic response to mTOR inhibitors.

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase

The C‐terminal domain of poly(A)‐binding protein (PABC) is a peptide‐binding domain found in poly(A)‐binding proteins (PABPs) and a HECT (homologous to E6‐AP C‐terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP‐interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP‐interacting protein‐1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of β‐turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein–peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull‐down assays. The results provide insight into the specificity of PABC in mediating PABP–protein interactions.

Molecular dissection of the eukaryotic initiation factor 4E (eIF4E) export-competent RNP

The eukaryotic translation initiation factor 4E (eIF4E) controls gene expression through its effects on mRNA export and cap‐dependent translation, both of which contribute to its oncogenic potential. In contrast to its translation function, the mRNA export function of eIF4E is poorly understood. Using an RNP isolation/mass spectrometry approach, we identified candidate cofactors of eIF4E mRNA export including LRPPRC. This protein associates with mRNAs containing the eIF4E‐sensitivity element (4E‐SE), and its overexpression alters the nuclear export of several eIF4E‐sensitive mRNAs. LRPPRC‐mediated alteration of eIF4Es mRNA export function requires the integrity of its eIF4E‐binding site and it coincides with the subcellular re‐distribution of eIF4E. The eIF4E export RNP is distinct in composition from the bulk mRNA export pathway, in that eIF4E‐ and eIF4E‐sensitive mRNAs do not associate with general mRNA export factors such as TAP/NXF1 or REF/Aly. Our data indicate that mRNA export pathways have evolved for specific mRNAs enabling the differential regulation of biochemical pathways by modulating the expression of groups of genes at the level of their export.

Poly(A) nuclease interacts with the C-terminal domain of polyadenylate-binding protein domain from poly(A)-binding protein.

The poly(A)-binding protein (PABP) is an essential protein found in all eukaryotes and is involved in an extensive range of cellular functions, including translation, mRNA metabolism, and mRNA export. Its C-terminal region contains a peptide-interacting PABC domain that recruits proteins containing a highly specific PAM-2 sequence motif to the messenger ribonucleoprotein complex. In humans, these proteins, including Paip1, Paip2, eRF3 (eukaryotic release factor 3), Ataxin-2, and Tob2, are all found to regulate translation through varying mechanisms. The following reports poly(A) nuclease (PAN) as a PABC-interacting partner in both yeast and humans. Their interaction is mediated by a PAM-2 motif identified within the PAN3 subunit. This site was identified in various fungal and animal species suggesting that the interaction is conserved throughout evolution. Our results indicate that PABP is directly involved in recruiting a deadenylase to the messenger ribonucleoprotein complex. This demonstrates a novel role for the PABC domain in mRNA metabolic processes and gives further insight into the function of PABP in mRNA maturation, export, and turnover.

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1

MicroRNAs (miRNAs) play critical roles in a variety of biological processes through widespread effects on protein synthesis. Upon association with the miRNA-induced silencing complex (miRISC), miRNAs repress target mRNA translation and accelerate mRNA decay. Degradation of the mRNA is initiated by shortening of the poly(A) tail by the CCR4-NOT deadenylase complex followed by the removal of the 5 cap structure and exonucleolytic decay of the mRNA. Here, we report a direct interaction between the large scaffolding subunit of CCR4-NOT, CNOT1, with the translational repressor and decapping activator protein, DDX6. DDX6 binds to a conserved CNOT1 subdomain in a manner resembling the interaction of the translation initiation factor eIF4A with eIF4G. Importantly, mutations that disrupt the DDX6-CNOT1 interaction impair miRISC-mediated gene silencing in human cells. Thus, CNOT1 facilitates recruitment of DDX6 to miRNA-targeted mRNAs, placing DDX6 as a downstream effector in the miRNA silencing pathway.

Cap‐free structure of eIF4E suggests a basis for conformational regulation by its ligands

The activity of the eukaryotic translation initiation factor eIF4E is modulated through conformational response to its ligands. For example, eIF4G and eIF4E‐binding proteins (4E‐BPs) modulate cap affinity, and thus physiological activity of eIF4E, by binding a site distal to the 7‐methylguanosine cap‐binding site. Further, cap binding substantially modulates eIF4Es affinity for eIF4G and the 4E‐BPs. To date, only cap‐bound eIF4E structures were reported. In the absence of structural information on the apo form, the molecular underpinnings of this conformational response mechanism cannot be established. We report here the first cap‐free eIF4E structure. Apo‐eIF4E exhibits structural differences in the cap‐binding site and dorsal surface relative to cap‐eIF4E. Analysis of structure and dynamics of apo‐eIF4E, and changes observed upon ligand binding, reveal a molecular basis for eIF4Es conformational response to these ligands. In particular, alterations in the S4‐H4 loop, distal to either the cap or eIF4G binding sites, appear key to modulating these effects. Mutation in this loop mimics these effects. Overall, our studies have important implications for the regulation of eIF4E.

A novel 4EHP-GIGYF2 translational repressor complex is essential for mammalian development

ABSTRACT The binding of the eukaryotic initiation factor 4E (eIF4E) to the mRNA 5′ cap structure is a rate-limiting step in mRNA translation initiation. eIF4E promotes ribosome recruitment to the mRNA. In Drosophila, the eIF4E homologous protein (d4EHP) forms a complex with binding partners to suppress the translation of distinct mRNAs by competing with eIF4E for binding the 5′ cap structure. This repression mechanism is essential for the asymmetric distribution of proteins and normal embryonic development in Drosophila. In contrast, the physiological role of the mammalian 4EHP (m4EHP) was not known. In this study, we have identified the Grb10-interacting GYF protein 2 (GIGYF2) and the zinc finger protein 598 (ZNF598) as components of the m4EHP complex. GIGYF2 directly interacts with m4EHP, and this interaction is required for stabilization of both proteins. Disruption of the m4EHP-GIGYF2 complex leads to increased translation and perinatal lethality in mice. We propose a model by which the m4EHP-GIGYF2 complex represses translation of a subset of mRNAs during embryonic development, as was previously reported for d4EHP.

Stability of Eukaryotic Translation Initiation Factor 4E mRNA Is Regulated by HuR, and This Activity Is Dysregulated in Cancer

ABSTRACT Eukaryotic translation initiation factor 4E (eIF4E) is encoded by a potent oncogene which is highly elevated in many human cancers. Few studies have investigated how the level, and thus activity, of eIF4E is regulated in healthy (noncancerous) cells and how they become elevated in malignant cells. Here, our studies reveal a novel mechanism by which eIF4E levels are regulated at the level of mRNA stability. Two factors known to modulate transcript stability, HuR and the p42 isoform of AUF1, compete for binding to the 3′ untranslated regions (3′UTRs) of eIF4E mRNAs. We identified a distinct AU-rich element in the 3′UTR of eIF4E which is responsible for HuR-mediated binding and stabilization. Our studies show that HuR is upregulated in malignant cancer specimens characterized by high eIF4E levels and that its depletion leads to reduction in eIF4E levels. Further, HuR and eIF4E regulate a common set of transcripts involved in cellular proliferation (cyclin D1 and c-myc) and neoangiogenesis (vascular endothelial growth factor), which suggests a functional connection between HuR and eIF4E in the regulation of these important processes. In summary, we present a novel model for the regulation of eIF4E expression and show that this model is relevant to elevation of eIF4E levels in malignant cells.

Signalling to eIF4E in cancer

Translational control plays a critical role in the regulation of gene expression in eukaryotes and affects many essential cellular processes, including proliferation, apoptosis and differentiation. Under most circumstances, translational control occurs at the initiation step at which the ribosome is recruited to the mRNA. The eukaryotic translation initiation factor 4E (eIF4E), as part of the eIF4F complex, interacts first with the mRNA and facilitates the recruitment of the 40S ribosomal subunit. The activity of eIF4E is regulated at many levels, most profoundly by two major signalling pathways: PI3K (phosphoinositide 3-kinase)/Akt (also known and Protein Kinase B, PKB)/mTOR (mechanistic/mammalian target of rapamycin) and Ras (rat sarcoma)/MAPK (mitogen-activated protein kinase)/Mnk (MAPK-interacting kinases). mTOR directly phosphorylates the 4E-BPs (eIF4E-binding proteins), which are inhibitors of eIF4E, to relieve translational suppression, whereas Mnk phosphorylates eIF4E to stimulate translation. Hyperactivation of these pathways occurs in the majority of cancers, which results in increased eIF4E activity. Thus, translational control via eIF4E acts as a convergence point for hyperactive signalling pathways to promote tumorigenesis. Consequently, recent works have aimed to target these pathways and ultimately the translational machinery for cancer therapy.