Amedee des Georges

Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit

Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5′-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5, 6, 7, 9, 10, 11, 12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S–IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.

High-Resolution Cryo-Electron Microscopy Structure of the Trypanosoma Brucei Ribosome.

Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.

Structure of mammalian eif3 in the context of the 43s preinitiation complex.

During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5′-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Å resolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.

Structure of the mammalian ribosomal pre-termination complex associated with eRF1•eRF3•GDPNP

Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3’s GTPase activity.

Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus

Background: The lipid and protein contributions to the mechanical properties of the influenza viral envelope are unknown. Results: The influenza viral envelope is 10 times softer than a viral protein-capsid coat but stiffer than a liposome. Conclusion: Membrane-associated proteins contribute to the mechanical stiffness of the viral envelope. Significance: The mechanical properties of the envelope are critical for the viral pH-regulated life cycle. The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm−1) than a viral protein-capsid coat and sustains deformations of one-third of the virions diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.

Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome

Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC βII binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map which shows that PKC βII interacts with RACK1, a seven-bladed β-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1. This study is a first step in understanding the molecular framework of PKC βII/RACK1 interaction and its role in translation.

High-resolution Cryo-EM Structure of the Trypanosoma brucei Ribosome: A Case Study

Single-particle cryo-electron microscopy has the immense advantage over crystallography in being able to image frozen-hydrated biological complexes in their “native” state, in solution. For years the ribosome has been the benchmark sample for particles without symmetry. It has witnessed steady improvement in resolution from the very first single-particle 3D reconstruction to today’s reconstructions at near-atomic resolution. In this study, we describe the different steps of sample preparation, data collection, data processing, and modeling that led to the 5A structure of the T. brucei ribosome [32]. A local resolution estimation demonstrates the extent to which resolution can be anisotropic and pinpoints regions of higher heterogeneity or structural flexibility. This study also shows an example of misuse of spatial frequency filters leading to overfitting of the data and the artifacts that can be observed in the resulting density map.

33 High-resolution cryo-EM structure of the Trypanosoma brucei 80S: a unique eukaryotic ribosome

Eukaryotic 80S ribosomes of known structure are far more complex than their 70S bacterial counterparts. Those from Saccharomyces cerevisiae, Tetrahymena thermophila, and Triticum aestivum, for example, bear insertions of ribosomal RNA (rRNA) called expansion segments (ES) and additional ribosomal proteins. The ribosomes of the kinetoplastid Trypanosoma brucei, though, are especially fascinating: structurally and their other kinetoplastids’ ribosomes bear very large ESs, as well as smaller ESs, and protein extensions. Additionally, T. brucei ribosomes require novel protein factors for maturation, although they do not require several eukaryotic initiation factors or a recycling factor. As a species, T. brucei is fascinating not only in terms of structure, but also in terms of gene expression and even public health: the species is responsible for the incurable, terminal human African Trypanosomiasis (sleeping sickness); and during post-transcriptional regulation, a single common RNA segment called a splice leader is trans-spliced onto the 5′ ends of many of T. brucei’s mRNAs. The purpose of this splice event in translation is unknown. Here, we present a high-resolution structure of the T. brucei ribosome which contributes a great deal to addressing the above unknowns. We have employed map segmentation, homology modeling, ab initio rRNA modeling, and Molecular Dynamics Flexible Fitting (MDFF) to model the ribosome’s atomic structure. The positions and structures of the ribosome’s novel ESs and protein extensions were previously unknown, but our structure reveals the precise spatial contexts of these components. With this information in hand, we can begin to decipher T. brucei’s unusual translational requirements.

ENRICH: a fast method to improve the quality of flexible macromolecular reconstructions

Cryo-electron microscopy using single particle analysis requires the computational averaging of thousands of projection images captured from identical macromolecules. However, macromolecules usually present some degree of flexibility showing different conformations. Computational approaches are then required to classify heterogeneous single particle images into homogeneous sets corresponding to different structural states. Nonetheless, sometimes the attainable resolution of reconstructions obtained from these smaller homogeneous sets is compromised because of reduced number of particles or lack of images at certain macromolecular orientations. In these situations, the current solution to improve map resolution is returning to the electron microscope and collect more data. In this work, we present a fast approach to partially overcome this limitation for heterogeneous data sets. Our method is based on deforming and then moving particles between different conformations using an optical flow approach. Particles are then merged into a unique conformation obtaining reconstructions with improved resolution, contrast and signal-to-noise ratio, then, partially circumventing many issues that impact obtaining high quality reconstructions from small data sets. We present experimental results that show clear improvements in the quality of obtained 3D maps, however, there are also limits to this approach, which we discuss in the manuscript.