48S Complex Research Articles

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, 5-7, 9-12), 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.

Molecular Composition and Ultrastructure of the Caveolar Coat Complex

Author SummaryCaveolae are flask-shaped invaginations in the plasma membrane of many mammalian cell types, and are particularly abundant in fat cells, muscle cells, and the cells that line blood vessels. Although caveolae are likely to be important for cellular responses to mechanical stress, intracellular trafficking, and signaling events, we still lack an understanding of the precise molecular mechanisms for how they form and carry out these functions. Here we address the question of how caveolae are made. Recent years have seen a considerable expansion of the catalogue of known protein components present in caveolae. Our study shows that the main protein components, cavins and caveolins, assemble into one specific complex. We reveal how different amounts of two caveolar proteins, cavin 2 and cavin 3, may be incorporated into this single type of complex, thereby potentially conferring different functional properties on caveolae. Using electron microscopy, we demonstrate that the protein complex is distributed all around the membrane bulb of caveolae, and so can be truly described as the caveolar coat. The caveolar coat excludes the protein EHD2, which regulates the dynamics of caveolae—this protein has a distinct distribution at the caveolar neck. These findings provide the basis for a more complete understanding of the network of protein interactions that produces caveolae.

Control of Translation and miRNA-Dependent Repression by a Novel Poly(A) Binding Protein, hnRNP-Q

Author SummaryThe regulation of mRNA translation and stability is of paramount importance for almost every cellular function. In eukaryotes, the poly(A) binding protein (PABP) is a central regulator of both global and mRNA-specific translation. PABP simultaneously interacts with the 3′ poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G). These interactions circularize the mRNA and stimulate translation. PABP also regulates specific mRNAs by promoting miRNA-dependent deadenylation and translational repression. A key step in understanding PABP's functions is to identify factors that affect its association with the poly(A) tail. Here we show that the cytoplasmic isoform of the mouse heterogeneous nuclear ribonucleoprotein Q (hnRNP-Q2/SYNCRIP), which exhibits binding preference to poly(A), interacts with the poly(A) tail by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to the poly(A) tail. Depleting hnRNP-Q2 stimulates translation in cell-free extracts and in cultured cells, in agreement with its function as translational repressor. In addition, hnRNP-Q2 impeded miRNA-mediated deadenylation and repression of target mRNAs, which requires PABP. Thus, competition from hnRNP-Q2 provides a novel mechanism by which multiple functions of PABP are regulated. This regulation could play important roles in various biological processes, such as development, viral infection, and human disease.

Listerin-Dependent Nascent Protein Ubiquitination Relies on Ribosome Subunit Dissociation

SummaryQuality control of defective mRNAs relies on their translation to detect the lesion. Aberrant proteins are therefore an obligate byproduct of mRNA surveillance and must be degraded to avoid disrupting protein homeostasis. These defective translation products are thought to be ubiquitinated at the ribosome, but the mechanism of ubiquitin ligase selectivity for these ribosomes is not clear. Here, we in vitro reconstitute ubiquitination of nascent proteins produced from aberrant mRNAs. Stalled 80S ribosome-nascent chain complexes are dissociated by the ribosome recycling factors Hbs1/Pelota/ABCE1 to a unique 60S-nascent chain-tRNA complex. The ubiquitin ligase Listerin preferentially recognizes 60S-nascent chains and triggers efficient nascent chain ubiquitination. Interfering with Hbs1 function stabilizes 80S complexes, precludes efficient Listerin recruitment, and reduces nascent chain ubiquitination. Thus, ribosome recycling factors control Listerin localization, explaining how translation products of mRNA surveillance are efficiently ubiquitinated while sparing translating ribosomes.

English

Introduction Organisms are able to generate organised responses to environmental changes by controlling the level of messenger RNA translation. The ability to respond rapidly allows the mobilization of the host’s defences and coordination of innate response to different infections. A large number of microbial pathogens inhibit the translation of host cells. Bacterial and virus infections interfere with protein synthesis of the host and activating or suppressing the innate responses, thus manipulating the translational machinery. Mortality, morbidity and economic adverse effects associated with fungal infections along with the emergence of resistance to antifungal agents make it necessary to gain a better understanding of the pathogenesis and discovery of new agents for treatment of those infections. There is little knowledge regarding the role of translational regulation in fungus–host relationship. The present review aims to show how translation regulation occurs in bacteria and virus infections and shows a mysterious role of translational regulation in the context of fungal infections. Conclusion In the last few years, incidence of fungal diseases has dramatically increased especially in immunodepressed individuals, representing a serious health problem since these diseases are associated with high mortality. There is a growing list of reports describing antifungal resistance in clinical isolates. Considering these facts, it is necessary for a better understanding of the pathophysiology of fungal infections that may reveal new therapeutic targets. Introduction The regulated translation of mRNAs in eukaryotes is a powerful tool of posttranscriptional control of gene expression. Translation has a fundamental role in cell growth and differentiation, development, learning, memory and synaptic plasticity. This process and its regulation have a relevant role in infections and host defence, allowing quick responses to contain pathogenic microorganisms. Regulation of these events occurs primarily at the steps of initiation and elongation. Figure 1 resumes the process. Before mRNA decoding, the eukaryotic 40S ribosomal subunit forms a 43S pre-initiation complex composed of different eukaryotic translation initiation factors (eIFs) and initiator tRNA charged with methionine (Met-tRNAi—see Figure 1). This association involves elF2, a heterotrimer that forms an active ternary complex (TC) containing GTP and aminoacylated initiator tRNA. In the presence of eIF4F complex, TC carries Met-tRNAi to 40S ribosome, a fundamental step to start protein synthesis at AUG codon or cognates1. eIF4F binds to the cap structure present in the 5 -end of eukaryotic mRNAs. This interaction promotes correct positioning of ribosome relative to mRNA, thus facilitating the ATP-dependent movement of it that is named as ‘scanning’ and locates the translation initiation codon AUG2. eIF4F is composed of a cap-binding protein (eIF4E), an RNA helicase (eIF4A) and a scavenger protein (eIF4G). eIF4E binds to the cap while eIF4G associates with eIF3 linked to a ribosome, allowing correct positioning of 43S complex at the mRNA 5 -end. Besides, eIF4E binds to PABP (poly(A)-binding protein) connecting 5 and 3 -ends of mRNAs, resulting in mRNA circularization that is supposed to be important for a rapid turnover in protein synthesis1. Translation initiation is the limiting step of protein synthesis in most circumstances, and then the process is subject to elegant regulatory mechanisms1. Initiation step is regulated by the availability of eIF4F2. The binding of eIF4E to eIF4G is modulated by a serine/threonine kinase, TOR1, which responds to growth factors, nutrients, amino acids, oxygen and energy availability3. In this context, TOR1 in a TORC1 complex, which controls eIF4E activity through phosphorylation of 4E-BPs (eIF4Ebinding proteins). Hyperphosphorylated 4E-BPs interact with eIF4E, preventing the formation of eIF4F complex and thus suppressing the cap-dependent translation, while hypophosphorylation of these proteins by mTORC1 promotes the release of eIF4E-eIF4G binding, stimulating the cap-dependent translation. After AUG initiation codon recognition, eIF5 stimulates the hydrolysis of GTP bound to eIF21. In a reaction catalysed by eIF5B, the 40S ribosomal subunit binds to 60S and * Corresponding author Email: gouveia_va@yahoo.com; gouveiava@ ufmg.br Laboratorio de Biologia Celular de Microrganismos, Departamento de Microbiologia – ICB, Universidade Federal de Minas Gerais, Brazil

Structure of the Mammalian Ribosomal 43S Preinitiation Complex Bound to the Scanning Factor DHX29

Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAi(Met)), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6Å resolution. It reveals that eIF2 interacts with the 40S subunit via its α subunit and supports Met-tRNAi(Met) in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.

Direct Monitoring of Initiation Factor Dynamics through Formation of 30S and 70S Translation-Initiation Complexes on a Quartz Crystal Microbalance

Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S-IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S-IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S-IC) and a 70S-IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27 MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N-terminally fused to biotin carboxyl carrier protein (bio-BCCP), were immobilized on a Neutravidin-covered QCM plate. By using bio-BCCP-IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S-IC, and formation of the 70S-IC. The kinetic parameters implied that the release of IF2 from the 70S-IC could be the rate-limiting step in translation initiation. The IF3-immobilized QCM revealed that the affinity of IF3 for the 30S complex decreased upon the addition of mRNA and fMet-tRNA(fMet) but did not lead to complete dissociation from the 30S-IC. These results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S-IC and 70S-IC and is finally induced to dissociate from ICs by 50S binding. This methodology demonstrated here is applicable to investigate the role of IFs in translation initiation driven by other pathways.

ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris)

Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S-13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.

Ribosomal Initiation Complex Assembly within the Wild-Strain of Coxsackievirus B3 and Live-Attenuated Sabin3-like IRESes during the Initiation of Translation

Coxsackievirus B3 (CVB3) is an enterovirus of the family of Picornaviridae. The Group B coxsackieviruses include six serotypes (B1 to B6) that cause a variety of human diseases, including myocarditis, meningitis, and diabetes. Among the group B, the B3 strain is mostly studied for its cardiovirulence and its ability to cause acute and persistent infections. Translation initiation of CVB3 RNA has been shown to be mediated by a highly ordered structure of the 5′-untranslated region (5′UTR), which harbors an internal ribosome entry site (IRES). Translation initiation is a complex process in which initiator tRNA, 40S and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of the mRNA. We have previously addressed the question of whether the attenuating mutations of domain V of the poliovirus IRES were specific for a given genomic context or whether they could be transposed and extrapolated to a genomic related virus, i.e., CVB3 wild-type strain. In this context, we have described that Sabin3-like mutation (U473→C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we analyzed the efficiency of formation of ribosomal initiation complexes 48S and 80S through 10%–30% and 10%–50% sucrose gradients using rabbit reticulocyte lysates (RRLs) and stage-specific translation inhibitors: 5′-Guanylyl-imidodiphosphate (GMP-PNP) and Cycloheximide (CHX), respectively. We demonstrated that the interaction of 48S and 80S ribosomal complexes within the mutant CVB3 RNA was abolished compared with the wild-type RNA by ribosome assembly analysis. Taken together, it is possible that the mutant RNA was unable to interact with some trans-acting factors critical for enhanced IRES function.

MicroRNAs Mediate Gene Silencing via Multiple Different Pathways in Drosophila

MicroRNAs (miRNAs) guide RNA-induced silencing complex (RISC) that contains an Argonaute family protein to complementary target messenger RNAs (mRNAs). Via RISC, miRNAs silence the expression of target mRNAs by shortening the poly(A) tail-which leads to mRNA decay-and by repressing translation. It has been suggested that GW182, an Argonaute-associating protein, plays the central role in such microRNA actions. Here we show that, although GW182 is obligatory for poly(A) shortening, translational repression by microRNAs occurs even in the absence of GW182. Yet, GW182 is also capable of inducing translational repression independently. Both of these translational repression mechanisms block formation of 48S and 80S ribosomal complexes. Thus microRNAs utilize at least three distinct silencing pathways: GW182-mediated deadenylation and GW182-dependent and -independent repression of early translation initiation. Differential contribution from these multiple pathways may explain previous, apparently contradictory observations of how microRNAs inhibit protein synthesis.

Severe Acute Respiratory Syndrome Coronavirus Protein nsp1 Is a Novel Eukaryotic Translation Inhibitor That Represses Multiple Steps of Translation Initiation

Severe acute respiratory syndrome (SARS) coronavirus nonstructural protein 1 (nsp1) binds to the 40S ribosomal subunit and inhibits translation, and it also induces a template-dependent endonucleolytic cleavage of host mRNAs. nsp1 inhibits the translation of cap-dependent and internal ribosome entry site (IRES)-driven mRNAs, including SARS coronavirus mRNAs, hepatitis C virus (HCV), and cricket paralysis virus (CrPV) IRES-driven mRNAs that are resistant to nsp1-induced RNA cleavage. We used an nsp1 mutant, nsp1-CD, lacking the RNA cleavage function, to delineate the mechanism of nsp1-mediated translation inhibition and identify the translation step(s) targeted by nsp1. nsp1 and nsp1-CD had identical inhibitory effects on mRNA templates that are resistant to nsp1-induced RNA cleavage, implying the validity of using nsp1-CD to dissect the translation inhibition function of nsp1. We provide evidence for a novel mode of action of nsp1. nsp1 inhibited the translation initiation step by targeting at least two separate stages: 48S initiation complex formation and the steps involved in the formation of the 80S initiation complex from the 48S complex. nsp1 had a differential, mRNA template-dependent, inhibitory effect on 48S and 80S initiation complex formation. nsp1 inhibited different steps of translation initiation on CrPV and HCV IRES, both of which initiate translation via an IRES-40S binary complex intermediate; nsp1 inhibited binary complex formation on CrPV IRES and 48S complex formation on HCV IRES. Collectively, the data revealed that nsp1 inhibited translation by exerting its effect on multiple stages of translation initiation, depending on the mechanism of initiation operating on the mRNA template.

The 70S ribosome modulates the ATPase activity of Escherichia coli YchF

YchF is one of two universally conserved GTPases with unknown cellular function. As a first step toward elucidating YchF’s cellular role, we performed a detailed biochemical characterization of the protein from Escherichia coli. Our data from fluorescence titrations not only confirmed the surprising finding that YchFE.coli binds adenine nucleotides more efficiently than guanine nucleotides, but also provides the first evidence suggesting that YchF assumes two distinct conformational states (ATP- and ADP-bound) consistent with the functional cycle of a typical GTPase. Based on an in vivo pull-down experiment using a His-tagged variant of YchF from E. coli (YchFE.coli), we were able to isolate a megadalton complex containing the 70S ribosome. Based on this finding, we report the successful reconstitution of a YchF•70S complex in vitro, revealing an affinity (KD) of the YchFE.coli•ADPNP complex for 70S ribosomes of 3 μM. The in vitro reconstitution data also suggests that the identity of the nucleotide-bound state of YchF (ADP or ATP) modulates its affinity for 70S ribosomes. A detailed Michaelis-Menten analysis of YchF’s catalytic activity in the presence and the absence of the 70S ribosome and its subunits revealed for the first time that the 70S ribosome is able to stimulate YchF’s ATPase activity (~10-fold), confirming the ribosome as part of the functional cycle of YchF. Our findings taken together with previously reported data for the human homolog of YchF (hOLA1) indicate a high level of evolutionary conservation in the enzymatic properties of YchF and suggest that the ribosome is the main functional partner of YchF not only in bacteria.

Eukaryotic initiation factor 5B: A new player for the anti-hepatitis C virus effect of ribavirin?

The addition of the broad-spectrum antiviral agent ribavirin (RBV), a synthetic guanosine analog, to interferon-alpha (IFNα) monotherapy has been a major breakthrough in the treatment of patients with hepatitis C virus (HCV), as it greatly improved treatment response rates. Although several mechanisms of action have been proposed for RBV’s antiviral activity, each with some experimental evidence, the precise mechanism by which it acts synergistically with IFNα has remained elusive. A cornerstone of the antiviral IFNα response is phosphorylation of the α subunit of eukaryotic initiation factor (eIF)2. This limits the availability of eIF2⋅GTP⋅Met-tRNAiMet ternary complexes, reduces formation of the 43S preinitiation complexes, ultimately blocking viral (and most cellular) mRNA translation. However recent studies indicated that translation driven by the HCV internal ribosome entry site (IRES) is insensitive to eIF2α phosphorylation. Particularly, in addition to the general eIF2-dependent pathway of translation, the HCV IRES makes use of a bacterial-like, eIF2-independent pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. Together, these observations support a model in which cellular stresses that induce eIF2α phosphorylation (e.g. treatment with IFNα) cause HCV IRES-directed translation to switch from an eIF2-dependent mode to an eIF5B-dependent mode, defining a tactic used by HCV to evade the INFα response. Eukaryotic eIF5B is a ribosome-dependent GTPase that is responsible for 80S complex formation in translation initiation but shows much lower affinities for GTP than to other GTPases, thus suggesting that it may mis-incorporate the RBV triphosphate (RTP) in place of GTP even at the RBV concentrations achieved in clinical use. Consequently, we theorize that RTP bound to eIF5B lowering its affinity for ribosome, blocks the 80S complex formation on HCV IRES inhibiting the eIF5B-dependent translation used by HCV to elude IFNα response. In conclusion, our hypothesis provides a mechanistic explanation for the phenomenon of RBV enhancement in INFα-based therapy.

Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.

Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits

In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of the cytoplasmic translation initiation factor eIF5b/Fun12. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associate with Fun12 and form 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP-hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together.

Heterogeneous pathways and timing of factor departure during translation initiation

Initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation1. Initiation involves factor-driven assembly at a start codon of an mRNA of an elongation competent 70S ribosomal particle (in bacteria) from separated 30S and 50S subunits and initiator tRNA. Here we establish by direct single-molecule tracking the timing of initiator tRNA, initiation factor 2 (IF2), and 50S subunit joining during initiation. Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition. IF2 accelerates 50S subunit joining, and stabilizes the assembled 70S complex. Transition to elongation is gated by the departure of IF2 after GTP hydrolysis, allowing efficient arrival of elongator tRNAs to the second codon presented in the aminoacyl-tRNA acceptor site. These experiments highlight the power of single-molecule approaches to delineate mechanism in complex multicomponent systems.

Polysome Preparation, RNA Isolation and Analysis.

During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosome can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.

Ribosome Fractionation in Yeast

This protocol describes yeast ribosome fractionation in the gradient of sucrose. During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then dissociates into the 40S and 60S subunits on termination. During elongation by one ribosome, further ribosomes can initiate translation on the same mRNA to form polysomes. The mass of each polysomal complex is determined primarily by the number of ribosomes it contains. Hence, the population of polysomes within the cell can be size-fractionated by sucrose density gradient centrifugation on the basis of the loading of ribosomes on the mRNA. Several compounds help to maintain or to disrupt the polysomes (Figure 1).

Requirement of rRNA Methylation for 80S Ribosome Assembly on a Cohort of Cellular Internal Ribosome Entry Sites

Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.

Archaeal Translation Initiation Factor aIF2 Can Substitute for Eukaryotic eIF2 in Ribosomal Scanning during Mammalian 48S Complex Formation

Heterotrimeric translation initiation factor (IF) a/eIF2 ( archaeal/ eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus solfataricus can substitute for its mammalian counterpart in the reconstitution of eukaryotic 48S initiation complexes from purified components. aIF2 is able to correctly place the initiator Met-tRNA i into the P-site of the 40S ribosomal subunit and accompany the entire set of eukaryotic translation IFs in the process of cap-dependent scanning and AUG codon selection. However, it seems to be unable to participate in the following step of ribosomal subunit joining. In accordance with this, aIF2 inhibits rather than stimulates protein synthesis in mammalian cell-free system. The ability of recombinant aIF2 protein to direct ribosomal scanning suggests that some archaeal mRNAs may utilize this mechanism during translation initiation.