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...

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...

The cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) uses an unusual mechanism of initiating translation, whereby the IRES occupies the P-site of the ribosome and the initiating tRNA enters the A-site. In vitro experiments have demonstrated that the CrPV IGR IRES is able to bind purified ribosomes and form 80S complexes capable of synthesizing small peptides in the absence of any translation initiation factors. These results suggest that initiation by this IRES is factor-independent. To determine whether the IGR IRES functions in the absence of initiation factors in vivo, we assayed IGR IRES activity in various yeast strains harboring mutations in canonical translation initiation factors. We used a dicistronic reporter assay in yeast to determine whether the CrPV IGR IRES is able to promote translation sufficient to support growth in the presence of various deletions or mutations in translation initiation factors. Using this assay, we have previously shown that the CrPV IGR IRES functions efficiently in yeast when ternary complexes (eIF2 d GTP d initiator tRNA met ) are reduced. Here, we demonstrate that the CrPV IGR IRES activity does not require the eukaryotic initiation factors eIF4G1 or eIF5B, and it is enhanced when eIF2B, the eIF3b subunit of eIF3, or eIF4E are impaired. Taken together, these data support a model in which the CrPV IGR IRES is capable of initiating protein synthesis in the absence of any initiation factors in vivo, and suggests that the CrPV IGR IRES initiates translation by directly recruiting the ribosomal subunits in vivo.

The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLVassociated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5=-untranslated region (5=UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5=UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCEThe mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene expression.