Stefano Marzi

RACK1 Controls IRES-Mediated Translation of Viruses

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

This study reveals novel insights into how Escherichia coli ribosomal protein S1 functions as an RNA chaperone on the ribosome, unfolding and positioning mRNAs for translation initiation.

The role of mRNA structure in translational control in bacteria

During the past few years, our knowledge on RNA-based regulation in many organisms has tremendously increased. In bacteria, although transcriptional regulatory proteins remain key players in gene regulation, a wide variety of post-transcriptional regulatory mechanisms discovered highlights the importance of the mRNA structure in the regulation of gene expression. RNA-dependent regulation largely contributes to rapidly adapt the bacterial metabolism in response to environmental changes, stress and in establishment of virulence. Bacteria exploit the extraordinary ability of mRNA to fold into different structures in response to various signals (environmental cues, ligand binding). Induced mRNA conformational rearrangements can potentially regulate transcription, translation and mRNA stability. The present review focuses on the structures of regulatory regions of mRNA that have evolved to permit productive interactions with trans-acting regulators, such as protein or non-coding RNAs. Finally, we describe how particular properties of these regulatory complexes regulate translation initiation.

Function and ribosomal localization of aIF6, a translational regulator shared by archaea and eukarya

The translation factor IF6 is shared by the Archaea and the Eukarya, but is not found in Bacteria. The properties of eukaryal IF6 (eIF6) have been extensively studied, but remain somewhat elusive. eIF6 behaves as a ribosome-anti-association factor and is involved in miRNA-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. Here we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homologue (aIF6). We find that aIF6 binds specifically to the 50S ribosomal subunits, hindering the formation of 70S ribosomes and strongly inhibiting translation. aIF6 is uniformly expressed along the cell cycle, but it is upregulated following both cold- and heat shock. The aIF6 ribosomal binding site lies in the middle of the 30-S interacting surface of the 50S subunit, including a number of critical RNA and protein determinants involved in subunit association. The data suggest that the IF6 protein evolved in the archaeal–eukaryal lineage to modulate translational efficiency under unfavourable environmental conditions, perhaps acquiring additional functions during eukaryotic evolution.

Ribosomal Initiation Complexes Probed by Toeprinting and Effect of trans-Acting Translational Regulators in Bacteria

Toeprinting was developed to study the formation of ribosomal initiation complexes in bacteria. This approach, based on the inhibition of reverse transcriptase elongation, was used to monitor the effect of ribosomal components and translational factors on the formation of the active ribosomal initiation complex. Moreover, this method offers an easy way to study in vitro how mRNA conformational changes alter ribosome binding at the initiation site. These changes can be induced either by environmental cues (temperature, ion concentration), or by the binding of metabolites, regulatory proteins, and trans-acting RNAs. An experimental guide is given to follow the different steps of the formation of ribosomal initiation complexes in Escherichia coli and Staphylococcus aureus, and to monitor the mechanism of action of several regulators on translation initiation in vitro. Protocols to prepare the ribosome and the subunits are also given for Thermus thermophilus, Staphylococcus aureus, and Escherichia coli.

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor.

Significance This work reports unique insights into IF2 function during eubacterial translation initiation by addressing the function of the N domain within the structure of the full-length factor in isolated form or ribosome bound, using crystallography, SAXS, cryo-EM, fast kinetics, and single molecule fluorescence. Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNAfMet positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.

Staphylococcus aureus RNAIII and Its Regulon Link Quorum Sensing, Stress Responses, Metabolic Adaptation, and Regulation of Virulence Gene Expression.

Staphylococcus aureus RNAIII is one of the main intracellular effectors of the quorum-sensing system. It is a multifunctional RNA that encodes a small peptide, and its noncoding parts act as antisense RNAs to regulate the translation and/or the stability of mRNAs encoding transcriptional regulators, major virulence factors, and cell wall metabolism enzymes. In this review, we explain how regulatory proteins and RNAIII are embedded in complex regulatory circuits to express virulence factors in a dynamic and timely manner in response to stress and environmental and metabolic changes.

Multiple ways to regulate translation initiation in bacteria: Mechanisms, regulatory circuits, dynamics.

To adapt their metabolism rapidly and constantly in response to environmental variations, bacteria often target the translation initiation process, during which the ribosome assembles on the mRNA. Here, we review different mechanisms of regulation mediated by cis-acting elements, sRNAs and proteins, showing, when possible, their intimate connection with the translational apparatus. Indeed the ribosome itself could play a direct role in several regulatory mechanisms. Different features of the regulatory signals (sequences, structures and their positions on the mRNA) are contributing to the large variety of regulatory mechanisms. Ribosome heterogeneity, variation of individual cells responses and the spatial and temporal organization of the translation process add more layers of complexity. This hampers to define manageable set of rules for bacterial translation initiation control.

RNA switches regulate initiation of translation in bacteria

Abstract A large variety of RNA-based mechanisms have been uncovered in all living organisms to regulate gene expression in response to internal and external changes, and to rapidly adapt cell growth in response to these signals. In bacteria, structural elements in the 5′ leader regions of mRNAs have direct effects on translation initiation of the downstream coding sequences. The docking and unfolding of these mRNAs on the 30S subunit are critical steps in the initiation process directly modulating and timing translation. Structural elements can also undergo conformational changes in response to environmental cues (i.e., temperature sensors) or upon binding of a variety of trans-acting factors, such as metabolites, non-coding RNAs or regulatory proteins. These RNA switches can temporally regulate translation, leading either to repression or to activation of protein synthesis.

Functional analysis of the translation factor aIF2/5B in the thermophilic archaeon Sulfolobus solfataricus

The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome‐dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell‐free protein‐synthesizing system. Moreover, the degree of functional conservation of the IF2‐like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of ‘chimeric’ proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.