Summary How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked Dyskeratosis Congenita (X-DC) and Hoyeraal-Hreidarsson syndrome (HH). Here we characterize ribosomes isolated from a yeast strain where Cbf5p, the yeast homologue of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A- and P-sites as well as the cricket paralysis virus IRES (Internal Ribosome Entry Site), which interacts with both the P- and E-sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans.

Most eukaryotic mRNAs are translated using a cap-dependent mechanism of translation. However, ;10% of mammalian mRNAs initiate translation using a cap-independent mechanism that is not well understood. These mRNAs contain an internal ribosome entry site (IRES) located in the 59 untranslated region. The cricket paralysis virus (CrPV) intergenic region IRES (IGR IRES) functions in yeast, mammals, and plants, and does not require any translation initiation factors. We used yeast genetics to understand how ribosomes are recruited directly to the mRNA by an IRES. We found that Rps25p has an essential role in CrPV IGR IRES activity in yeast and mammalian cells but not in cap-dependent translation. Purified 40S ribosomal subunits lacking Rps25 are unable to bind to the IGR IRES in vitro. The hepatitis C virus (HCV) IRES also requires Rps25, demonstrating the function of Rps25 is conserved across IRES types. Yeast strains lacking Rps25 exhibit only slight defects in global translation, readthrough, ribosome biogenesis, and programmed ribosomal frameshifting. This work is the first demonstration of a ribosomal protein that is specifically required for IRES-mediated translation initiation. Our findings provide us with the beginnings of a model for the molecular interactions of an IRES with the ribosome. Protein synthesis in eukaryotes is highly regulated both globally and in an mRNA-specific manner. The vast majority of eukaryotic mRNAs are translated in a capdependent manner, which requires multiple initiation factors to recruit the 40S ribosomal subunit to the 59 end of the message. Briefly, eIF4A, eIF4G, and eIF4E bind to the 59 m7 GpppN cap structure and recruit the 43S preinitiation complex, which consists of eIF3, eIF1, eIF1A, eIF5, and the ternary complex (Met-tRNA i , eIF2, and GTP) bound to the 40S small ribosomal subunit. The 48S preinitiation complex then scans the mRNA in the 59-to-39 direction until the AUG start codon is positioned in the peptidyl site (P site) of the 40S ribosomal subunit. At this point, GTP hydrolysis is triggered and eIF2 is released, along with other initiation factors. Then, eIF5B facilitates joining of the 60S ribosomal subunit and the second GTP is hydrolyzed to transition into the elongation phase. Under various cellular stresses or during viral infection, cap-dependent translation is globally repressed, and mRNAs that contain internal ribosome entry sites (IRESs) can be translated using a cap-independent mechanism of initiation (Sonenberg and Hinnebusch 2009). IRESs were originally discovered in picornaviruses 20 years ago, and since then they have been found in numerous other viral and cellular mRNAs (Bushell and Sarnow 2002;Spriggs et al. 2008). Viral IRESs can be generally grouped into four categories based on the number of canonical initiation factors and IRES trans-acting factors (ITAFs) that they require, as well as the placement and codon usage of the start site. Type I picornavirus IRESs require ITAFs, several canonical initiation factors, and initiator Met-tRNA i , and have...

As obligate intracellular parasites, virus reproduction requires host cell functions. Despite variations in genome size and configuration, nucleic acid composition, and their repertoire of encoded functions, all viruses remain unconditionally dependent on the protein synthesis machinery resident within their cellular hosts to translate viral messenger RNAs (mRNAs). A complex signaling network responsive to physiological stress, including infection, regulates host translation factors and ribosome availability. Furthermore, access to the translation apparatus is patrolled by powerful host immune defenses programmed to restrict viral invaders. Here, we review the tactics and mechanisms used by viruses to appropriate control over host ribosomes, subvert host defenses, and dominate the infected cell translational landscape. These not only define aspects of infection biology paramount for virus reproduction, but continue to drive fundamental discoveries into how cellular protein synthesis is controlled in health and disease.

bDuring viral infection or cellular stress, cap-dependent translation is shut down. Proteins that are synthesized under these conditions use alternative mechanisms to initiate translation. This study demonstrates that at least two alternative translation initiation routes, internal ribosome entry site (IRES) initiation and ribosome shunting, rely on ribosomal protein S25 (RPS25). This suggests that they share a mechanism for initiation that is not employed by cap-dependent translation, since cap-dependent translation is not affected by the loss of RPS25. Furthermore, we demonstrate that viruses that utilize an IRES or a ribosome shunt, such as hepatitis C virus, poliovirus, or adenovirus, have impaired amplification in cells depleted of RPS25. In contrast, viral amplification of a virus that relies solely on cap-dependent translation, herpes simplex virus, is not hindered. We present a model that explains how RPS25 can be a nexus for multiple alternative translation initiation pathways.T he predominant translation initiation pathway for cellular mRNAs is cap dependent, which requires recognition of the mRNA 5= cap structure by the cap binding protein eukaryotic initiation factor 4E (eIF4E). eIF4E interacts with a complex of eukaryotic initiation factors to recruit the ribosome to the 5= end of the mRNA (1). However, some viral and cellular mRNAs contain an internal ribosome entry site (IRES) in their 5= untranslated region (UTR) that recruits the ribosome in a cap-independent manner. This allows translation of key regulatory proteins under conditions where cap-dependent translation is downregulated, such as during cell stress (2). In fact, 5 to 10% of cellular mRNAs have been predicted to contain IRES elements. IRESs are enriched in genes that encode proteins regulating growth, differentiation, and responses to stress (3, 4). Viruses have coopted this pathway by inhibiting cap-dependent translation and using an IRES to recruit host ribosomes to synthesize viral proteins (5).The mechanism of IRES-mediated translation is not well understood; however, our best understanding has come from studying viral IRESs. Viral IRESs have been characterized functionally according to the type and number of initiation factors required (6). The picornaviral IRESs require several canonical initiation factors and noncanonical IRES trans-acting factors (ITAFs) (6, 7). The hepatitis C virus (HCV) IRES can bind directly to the 40S ribosomal subunit but requires additional factors to initiate protein synthesis (8-10). The most streamlined IRESs are found in the intergenic region (IGR) of the Dicistroviridae virus family; they recruit the 40S and 60S subunits to form functional 80S complexes in the absence of any initiation factors (11-13).The IGR IRES binds to the intersubunit surface of the 40S subunit and occupies the peptidyl (P) and exit (E) sites, whereas the HCV IRES binds to the solvent side of the 40S subunit with only the finger-like domain IIb occupying the E site (14-16). While the binding of these two IRESs to the 40S subuni...

Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S. cerevisiae, thereby conferring uracilindependent growth. Curiously, the IRES functions poorly in wildtype yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA met genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP͞ initiator tRNA met complexes. Furthermore, tRNA met -independent initiation was also observed in translation-competent extracts prepared from S. cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S. cerevisiae in the absence of eIF2 and initiator tRNA met , by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry. Saccharomyces cerevisiae has been an invaluable tool in the study of mechanisms of cap-dependent translation initiation (1, 2). However, efforts to use yeast as a model system to study the mechanism of cap-independent internal initiation of translation have been hampered by the absence of functional internal ribosome entry site (IRES) elements that can direct the synthesis of selectable marker gene products. The well-studied viral IRES elements located in the RNA genomes of encephalomyocarditis virus, poliovirus, and hepatitis C virus do not function in living S. cerevisiae (3-5). The reasons for these IRES elements being inactive in yeast remain unclear. In the case of poliovirus and hepatitis C virus, a small inhibitor RNA has been detected, and it has been postulated that this inhibitor RNA sequesters factors that are needed for IRES-mediated translation (4, 5).We have expressed approximately two million dicistronic mRNA species in yeast, containing unique nucleotide sequences in the intercistronic spacer. However, none of those sequences functioned as an IRES to mediate translation of the second cistron (unpublished observation). This finding was surprising because similar strategies have identified new synthetic IRES elements in mammalian cells (6, 7). The most likely reason why the yeast translation apparatus favors 5Ј end-mediated translation over internal initiation is the synergy by which the 5Ј terminal cap structure and the 3Ј terminal polyadenosine sequences direct the binding of ribosomal subunits to the 5Ј end of the ...