Crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5.
ABSTRACT: Death-associated protein 5 (DAP5) is a member of the eIF4G family of scaffolding proteins that mediate cap-independent translation initiation by recruiting the translational machinery to internal ribosomal entry sites (IRESs) on mRNA. The MIF4G domain of DAP5 directly interacts with the eukaryotic initiation factors eIF4A and eIF3 and enhances the translation of several viral and cellular IRESs. Here, the crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5 is presented.
Project description:Death-associated protein 5 (DAP5/p97) is a homolog of the eukaryotic initiation factor 4G (eIF4G) that promotes the IRES-driven translation of multiple cellular mRNAs. Central to its function is the middle domain (MIF4G), which recruits the RNA helicase eIF4A. The middle domain of eIF4G consists of tandem HEAT repeats that coalesce to form a solenoid-type structure. Here, we report the crystal structure of the DAP5 MIF4G domain. Its overall fold is very similar to that of eIF4G; however, significant conformational variations impart distinct surface properties that could explain the observed differences in IRES binding between the two proteins. Interestingly, quantitative analysis of the DAP5-eIF4A interaction using isothermal titration calorimetry reveals a 10-fold lower affinity than with the eIF4G-eIF4A interaction that appears to affect their ability to stimulate eIF4A RNA unwinding activity in vitro. This difference in stability of the complex may have functional implications in selecting the mode of translation initiation.
Project description:Kinetoplastida, a class of early-diverging eukaryotes that includes pathogenic Trypanosoma and Leishmania species, display key differences in their translation machinery compared with multicellular eukaryotes. One of these differences involves a larger number of genes encoding eIF4E and eIF4G homologs and the interaction pattern between the translation initiation factors. eIF4G is a scaffold protein which interacts with the mRNA cap-binding factor eIF4E, the poly(A)-binding protein, the RNA helicase eIF4A and the eIF3 complex. It contains the so-called middle domain of eIF4G (MIF4G), a multipurpose adaptor involved in different protein-protein and protein-RNA complexes. Here, the crystal structure of the MIF4G domain of T. cruzi EIF4G5 is described at 2.4?Å resolution, which is the first three-dimensional structure of a trypanosomatid MIF4G domain to be reported. Structural comparison with IF4G homologs from other eukaryotes and other MIF4G-containing proteins reveals differences that may account for the specific interaction mechanisms of MIF4G despite its highly conserved overall fold.
Project description:Viral internal ribosomal entry sites (IRESs) mediate end-independent translation initiation. There are 4 major structurally-distinct IRES groups: type 1 (e.g., poliovirus) and type 2 (e.g., encephalomyocarditis virus), which are dissimilar except for a Yn-Xm-AUG motif at their 3' borders, type 3 (e.g., hepatitis C virus), and type 4 (dicistroviruses). Type 2-4 IRESs mediate initiation by distinct mechanisms that are nevertheless all based on specific noncanonical interactions with canonical components of the translation apparatus, such as eukaryotic initiation factor (eIF) 4G (type 2), 40S ribosomal subunits (types 3 and 4), and eIF3 (type 3). The mechanism of initiation on type 1 IRESs is unknown. We now report that domain V of type 1 IRESs, which is adjacent to the Yn-Xm-AUG motif, specifically interacts with the central domain of eIF4G. The position and orientation of eIF4G relative to the Yn-Xm-AUG motif is analogous in type 1 and 2 IRESs. eIF4G promotes recruitment of eIF4A to type 1 IRESs, and together, eIF4G and eIF4A induce conformational changes at their 3' borders. The ability of mutant type 1 IRESs to bind eIF4G/eIF4A correlated with their translational activity. These characteristics parallel the mechanism of initiation on type 2 IRESs, in which the key event is binding of eIF4G to the J-K domain adjacent to the Yn-Xm-AUG motif, which is enhanced by eIF4A. These data suggest that fundamental aspects of the mechanisms of initiation on these unrelated classes of IRESs are similar.
Project description:Apoptosis is characterized by a translation switch from cap-dependent to internal ribosome entry site (IRES)-mediated protein translation. During apoptosis, several members of the eukaryotic initiation factor (eIF)4G family are cleaved specifically by caspases. Here we investigated which of the caspase-cleaved eIF4G family members could support cap-independent translation through IRES elements that retain activity in the dying cell. We focused on two major fragments arising from the cleavage of eIF4GI and death-associated protein 5 (DAP5) proteins (eIF4GI M-FAG/p76 and DAP5/p86, respectively), because they are the only potential candidates to preserve the minimal scaffold function needed to mediate translation. Transfection-based experiments in cell cultures indicated that expression of DAP5/p86 in cells stimulated protein translation from the IRESs of c-Myc, Apaf-1, DAP5, and XIAP. In contrast, these IRESs were refractory to the ectopically expressed eIF4GI M-FAG/p76. Furthermore, our study provides in vivo evidence that the caspase-mediated removal of the C-terminal tail of DAP5/p97 relieves an inhibitory effect on the protein's ability to support cap-independent translation through the DAP5 IRES. Altogether, the data suggest that DAP5 is a caspase-activated translation factor that mediates translation through a repertoire of IRES elements, supporting the translation of apoptosis-related proteins.
Project description:Picornavirus Type 1 IRESs comprise five principal domains (dII-dVI). Whereas dV binds eIF4G, a conserved AUG in dVI was suggested to stimulate attachment of 43S ribosomal preinitiation complexes, which then scan to the initiation codon. Initiation on Type 1 IRESs also requires IRES trans-acting factors (ITAFs), and several candidates have been proposed. Here, we report the in vitro reconstitution of initiation on three Type 1 IRESs: poliovirus (PV), enterovirus 71 (EV71), and bovine enterovirus (BEV). All of them require eIF2, eIF3, eIF4A, eIF4G, eIF4B, eIF1A, and a single ITAF, poly(C) binding protein 2 (PCBP2). In each instance, initiation starts with binding of eIF4G/eIF4A. Subsequent recruitment of 43S complexes strictly requires direct interaction of their eIF3 constituent with eIF4G. The following events can differ between IRESs, depending on the stability of dVI. If it is unstructured (BEV), all ribosomes scan through dVI to the initiation codon, requiring eIF1 to bypass its AUG. If it is structured (PV, EV71), most initiation events occur without inspection of dVI, implying that its AUG does not determine ribosomal attachment.
Project description:Translation initiation in eukaryotes is facilitated by the cap structure, m7GpppN (where N is any nucleotide). Eukaryotic translation initiation factor 4F (eIF4F) is a cap binding protein complex that consists of three subunits: eIF4A, eIF4E and eIF4G. eIF4G interacts directly with eIF4E and eIF4A. The binding site of eIF4E resides in the N-terminal third of eIF4G, while eIF4A and eIF3 binding sites are present in the C-terminal two-thirds. Here, we describe a new eukaryotic translational regulator (hereafter called p97) which exhibits 28% identity to the C-terminal two-thirds of eIF4G. p97 mRNA has no initiator AUG and translation starts exclusively at a GUG codon. The GUG-initiated open reading frame (907 amino acids) has no canonical eIF4E binding site. p97 binds to eIF4A and eIF3, but not to eIF4E. Transient transfection experiments show that p97 suppresses both cap-dependent and independent translation, while eIF4G supports both translation pathways. Furthermore, inducible expression of p97 reduces overall protein synthesis. These results suggest that p97 functions as a general repressor of translation by forming translationally inactive complexes that include eIF4A and eIF3, but exclude eIF4E.
Project description:Cadicivirus (CDV) is unique amongst picornaviruses in having a dicistronic genome with internal ribosomal entry sites (IRESs) preceding both open reading frames. Here, we investigated initiation on the 5'-terminal IRES. We report that the 982-nt long 5'UTR comprises 12 domains (d1-d12), five of which (d8-d12, nts 341-950) constitute a divergent Type I IRES. It comprises central elements (the apex of d10, d11 and the following polypyrimidine tract) that are homologous to corresponding elements in canonical Type 1 IRESs, and non-canonical flanking domains (d8, d9 and d12). In vitro reconstitution revealed that as with canonical Type I IRESs, 48S complex formation requires eukaryotic initiation factors (eIFs) 1, 1A, 2, 3, 4A, 4B and 4G, and the poly(C) binding protein 2 (PCBP2), and starts with specific binding of eIF4G/eIF4A to d11. However, in contrast to canonical Type I IRESs, subsequent recruitment of 43S ribosomal complexes does not require direct interaction of their eIF3 constituent with the IRES-bound eIF4G. On the other hand, the CDV IRES forms a 40S/eIF3/IRES ternary complex, with multiple points of contact. These additional interactions with translational components could potentially stimulate recruitment of the 43S complex and alleviate the necessity for direct eIF4G/eIF3 interaction.
Project description:Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap-binding complex and the 40S subunit. A discrete ?90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a fluorescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site-specific cross-linking approach, we unexpectedly show that the eIF4G-binding surface in eIF3 is comprised of the -c, -d and -e subunits. Screening multiple cross-linker positions reveals that eIF4G contains two distinct eIF3-binding subdomains within the previously identified eIF3-binding domain. Finally, by employing an eIF4G-dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3-eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome.
Project description:Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5'UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2? and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.
Project description:DEAD-box proteins are involved in all aspects of RNA processing. They bind RNA in an ATP-dependent manner and couple ATP hydrolysis to structural and compositional rearrangements of ribonucleoprotein particles. Conformational control is a major point of regulation for DEAD-box proteins to act on appropriate substrates and in a timely manner in vivo. Binding partners containing a middle domain of translation initiation factor 4G (MIF4G) are emerging as important regulators. Well-known examples are eIF4G and Gle1, which bind and activate the DEAD-box proteins eIF4A and Dbp5. Here, we report the mechanism of an inhibiting MIF4G domain. We determined the 2.0-Å resolution structure of the complex of human eIF4AIII and the MIF4G domain of the splicing factor Complexed With Cef1 (CWC22), an essential prerequisite for exon junction complex assembly by the splicing machinery. The CWC22 MIF4G domain binds both RecA domains of eIF4AIII. The mode of RecA2 recognition is similar to that observed in the activating complexes, yet is specific for eIF4AIII. The way the CWC22 MIF4G domain latches on the eIF4AIII RecA1 domain is markedly different from activating complexes. In the CWC22-eIF4AIII complex, the RNA-binding and ATP-binding motifs of the two RecA domains do not face each other, as would be required in the active state, but are in diametrically opposite positions. The binding mode of CWC22 to eIF4AIII reveals a facet of how MIF4G domains use their versatile structural frameworks to activate or inhibit DEAD-box proteins.