Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus

Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5' noncoding region called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of approximately 25 kDa in UV cross-linking experiments. In the present study, we examined the approximately 25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.     

Conserved functional domains and a novel tertiary interaction near the pseudoknot drive translational activity of hepatitis C virus and hepatitis C virus-like internal ribosome entry sites

The translational activity of the hepatitis C virus (HCV) internal ribosome entry site (IRES) and other HCV-like IRES RNAs depends on structured RNA elements in domains II and III, which serve to recruit the ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3 and the ternary eIF2/Met-tRNA_i^Met/GTP complex and subsequently domain II assists subunit joining. Porcine teschovirus-1 talfan (PTV-1) is a member of the Picornaviridae family, with a predicted HCV-like secondary structure, but only stem-loops IIId and IIIe in the 40S-binding domain display significant sequence conservation with the HCV IRES. Here, we use chemical probing to show that interaction sites with the 40S subunit and eIF3 are conserved between HCV and HCV-like IRESs. In addition, we reveal the functional role of a strictly conserved co-variation between a purine–purine mismatch near the pseudoknot (A–A/G) and the loop sequence of domain IIIe (GAU/CA). These nucleotides are involved in a tertiary interaction, which serves to stabilize the pseudoknot structure and correlates with translational efficiency in both the PTV-1 and HCV IRES. Our data demonstrate conservation of functional domains in HCV and HCV-like IRESs including a more complex structure surrounding the pseudoknot than previously assumed.     

Structural basis for the binding of IRES RNAs to the head of the ribosomal 40S subunit

Some viruses exploit internal initiation for their propagation in the host cell. This type of initiation is facilitated by structured elements (internal ribosome entry site, IRES) upstream of the initiator AUG and requires only a reduced number of canonical initiation factors. An important example are IRES of the virus family Dicistroviridae that bind to the inter-subunit side of the small ribosomal 40S subunit and lead to the formation of elongation-competent 80S ribosomes without the help of any initiation factor. Here, we present a comprehensive functional and structural analysis of eukaryotic-specific ribosomal protein rpS25 in the context of this type of initiation and propose a structural model explaining the essential involvement of rpS25 for hijacking the ribosome.     

Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction

Translation of the hepatitis C virus genomic RNA is mediated by an internal ribosome entry site (IRES). The 330-nt IRES RNA forms a binary complex with the small 40S ribosomal subunit as a first step in translation initiation. Here chemical probing and 4-thiouridine-mediated crosslinking are used to characterize the interaction of the HCV IRES with the HeLa 40S subunit. No IRES-18S rRNA contacts were detected, but several specific crosslinks to 40S ribosomal proteins were observed. The identity of the crosslinked proteins agrees well with available structural information and provides new insights into HCV IRES function. The protein-rich surface of the 40S subunit thus mediates the IRES-ribosome interaction.     

A central fragment of ribosomal protein S26 containing the eukaryote-specific motif YxxPKxYxK is a key component of the ribosomal binding site of mRNA region 5' of the E site codon

The eukaryotic ribosomal protein S26e (rpS26e) lacking eubacterial counterparts is a key component of the ribosomal binding site of mRNA region 5' of the codon positioned at the exit site. Here, we determined the rpS26e oligopeptide neighboring mRNA on the human 80S ribosome using mRNA analogues bearing perfluorophenyl azide-derivatized nucleotides at designed locations. The protein was cross-linked to mRNA analogues in specific ribosomal complexes, in which the derivatized nucleotide was located at positions -3 to -9. Digestion of cross-linked rpS26e with various specific proteolytic agents followed by identification of the resulting modified oligopeptides made it possible to map the cross-links to fragment 60-71. This fragment contains the motif YxxPKxYxK conserved in eukaryotic but not in archaeal rpS26e. Analysis of X-ray structure of the Tetrahymena thermophila 40S subunit showed that this motif is not implicated in the intraribosomal interactions, implying its involvement in translation process in a eukaryote-specific manner. Comparison of the results obtained with data on positioning of ribosomal ligands on the 40S subunit lead us to suggest that this motif is involved in interaction with both the 5'-untranslated region of mRNA and the initiation factor eIF3 specific for eukaryotes, providing new insights into molecular mechanisms of translation in eukaryotes.     

HCV and CSFV IRES domain II mediate eIF2 release during 80S ribosome assembly

Internal ribosome entry site (IRES) RNAs from the hepatitis C virus (HCV) and classical swine fever virus (CSFV) coordinate cap-independent assembly of eukaryotic 48S initiation complexes, consisting of the 40S ribosomal subunit, eukaryotic initiation factor (eIF) 3 and the eIF2/GTP/Met-tRNA(i)(Met) ternary complex. Here, we report that these IRESes also play a functional role during 80S ribosome assembly downstream of 48S complex formation, in promoting eIF5-induced GTP hydrolysis and eIF2/GDP release from the initiation complex. We show that this function is encoded in their independently folded IRES domain II and that it depends both on its characteristic bent conformation and two conserved RNA motifs, an apical hairpin loop and a loop E. Our data suggest a general mode of subunit joining in HCV and HCV-like IRESes.     

HCV IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation

Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES–rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.     

Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit

The molecular environment of the internal ribosome entry site (IRES element) of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group at nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the subunit between the head and the body aside the “beak” near the exit from the mRNA-binding channel.     

An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus

Hepatitis C virus translation is initiated on a approximately 330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5' end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG(266-268) in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.     

Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit

The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the outer subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.     

The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.     

Mechanism of ribosome recruitment by hepatitis C IRES RNA

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5' untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.     

Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome

The 5′-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.     

The pathway of HCV IRES-mediated translation initiation

The HCV internal ribosome entry site (IRES) directly regulates the assembly of translation initiation complexes on viral mRNA by a sequential pathway that is distinct from canonical eukaryotic initiation. The HCV IRES can form a binary complex with an eIF-free 40S ribosomal subunit. Next, a 48S-like complex assembles at the AUG initiation codon upon association of eIF3 and ternary complex. 80S complex formation is rate limiting and follows the GTP-dependent association of the 60S subunit. Efficient assembly of the 48S-like and 80S complexes on the IRES mRNA is dependent upon maintenance of the highly conserved HCV IRES structure. This revised model of HCV IRES translation initiation provides a context to understand the function of different HCV IRES domains during translation initiation.     

Crystal Structure of the Yeast Ribosomal Protein rpS3 in Complex with Its Chaperone Yar1

Eukaryotic ribosome assembly involves a plethora of factors, which ensure that a correctly folded ribosome contains all ribosomal protein components. Among these assembly factors, Yar1 has recently emerged as a molecular chaperone for ribosomal protein rpS3 of the small ribosomal subunit (40S) in yeast. In complex with its chaperone, rpS3 is imported into the nucleus and protected from aggregation. How rpS3 and other ribosomal proteins are initially sequestered and subsequently integrated into pre-ribosomal particles is currently poorly understood. Here, we present the crystal structure of yeast rpS3 in complex with its chaperone Yar1 at 2.8 Å resolution. The crystal structure rationalizes how Yar1 can protect rpS3 from aggregation while facilitating nuclear import and suggests a mechanism for a stepwise exchange of molecular partners that ribosomal proteins interact with during ribosome assembly.     

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit''s decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.     

Structural motifs of the bacterial ribosomal proteins S20, S18 and S16 that contact rRNA present in the eukaryotic ribosomal proteins S25, S26 and S27A,respectively

The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively.