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.

Programmed –1 ribosomal frameshift (–1 PRF) signals redirect translating ribosomes to slip back one base on messenger RNAs. Although well characterized in viruses, how these elements may regulate cellular gene expression is not understood. Here we describe a –1 PRF signal in the human mRNA encoding CCR5, the HIV-1 co-receptor. CCR5 mRNA-mediated –1 PRF is directed by an mRNA pseudoknot, and is stimulated by at least two microRNAs. Mapping the mRNA–miRNA interaction suggests that formation of a triplex RNA structure stimulates –1 PRF. A –1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay pathway. At least one additional mRNA decay pathway is also involved. Functional –1 PRF signals that seem to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors, suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells.

During cap-dependent translation of eukaryotic mRNAs, initiation factors interact with the 59 cap to attract ribosomes. When animal viruses translate in a cap-independent fashion, ribosomes assemble upstream of initiation codons at internal ribosome entry sites (IRES). In contrast, many plant viral genomes do not contain 59 ends with substantial IRES activity but instead have 39 translational enhancers that function by an unknown mechanism. A 393-nucleotide (nt) region that includes the entire 39 UTR of the Turnip crinkle virus (TCV) synergistically enhances translation of a reporter gene when associated with the TCV 59 UTR. The major enhancer activity was mapped to an internal region of ;140 nt that partially overlaps with a 100-nt structural domain previously predicted to adopt a form with some resemblance to a tRNA, according to a recent study by J.C. McCormack and colleagues. The T-shaped structure binds to 80S ribosomes and 60S ribosomal subunits, and binding is more efficient in the absence of surrounding sequences and in the presence of a pseudoknot that mimics the tRNA-acceptor stem. Untranslated TCV satellite RNA satC, which contains the TCV 39 end and 6-nt differences in the region corresponding to the T-shaped element, does not detectably bind to 80S ribosomes and is not predicted to form a comparable structure. Binding of the TCV T-shaped element by 80S ribosomes was unaffected by salt-washing, reduced in the presence of AcPhe-tRNA, which binds to the P-site, and enhanced binding of Phe-tRNA to the ribosome A site. Mutations that reduced translation in vivo had similar effects on ribosome binding in vitro. This strong correlation suggests that ribosome entry in the 39 UTR is a key function of the 39 translational enhancer of TCV and that the T-shaped element contains some tRNA-like properties.

There is something special about mRNA pseudoknots that allows them to elicit efficient levels of programmed −1 ribosomal frameshifting. Here, we present a synthesis of recent crystallographic, molecular, biochemical, and genetic studies to explain this property. Movement of 9 Å by the anticodon loop of the aminoacyl-tRNA at the accommodation step normally pulls the downstream mRNA a similar distance along with it. We suggest that the downstream mRNA pseudoknot provides resistance to this movement by becoming wedged into the entrance of the ribosomal mRNA tunnel. These two opposing forces result in the creation of a local region of tension in the mRNA between the A-site codon and the mRNA pseudoknot. This can be relieved by one of two mechanisms; unwinding the pseudoknot, allowing the downstream region to move forward, or by slippage of the proximal region of the mRNA backwards by one base. The observed result of the latter mechanism is a net shift of reading frame by one base in the 5 direction, that is, a −1 ribosomal frameshift.Keywords: Virus; ribosome; translation; genetic code; recoding; structure/function After a generation spent in the shadows, the ribosome is enjoying a renaissance. Recent breakthroughs in X-ray crystallography and cryoelectron microscopy have given us atomic-level views of this complex molecular machine Wimberly et al. 2000;Harms et al. 2001;Spahn et al. 2001;) that are bringing into focus the relationship between ribosome structure and function (Gabashvili et al. 1999;Agrawal et al. 2000;Carter et al. 2000;Frank and Agrawal 2000;Mueller et al. 2000;Nissen et al. 2000;Schluenzen et al. 2000;Beckmann et al. 2001;Nissen et al. 2001; Pioletti et al. 2001;Polacek et al. 2001;Thompson et al. 2001;Yusupova et al. 2001;Noller et al. 2002;Schmeing et al. 2002;Simonson and Lake 2002). One of the major requirements of the ribosome is to maintain translational reading frame, and an increasing number of cis-acting mRNA signals that alter this have been used to probe this essential function of the translational machinery. These translational "recoding" events (Gesteland and Atkins 1996) can take many forms, for example, "slips" of one or more bases, "hops" spanning as many as 50 nucleotides, and "shunts" around large mRNA secondary structures (for review, see Jacks 1990;Brierley 1995;Farabaugh 1996;Giedroc et al. 2000). Programmed −1 ribosomal frameshifting (−1 PRF) is the most widely used translational recoding mechanism of RNA viruses. The −1 PRF signal can be broken down into three discrete parts: the "slippery site", a linker region, and a downstream region of secondary mRNA structure, typically an mRNA pseudoknot. Mutagenesis studies from many different laboratories have demonstrated that the primary sequence of the slippery site and its placement in relation to the incoming translational reading frame is critical: It must be X XXY YYZ, where X must be a stretch of three identical nucleotides, Y is either AAA or UUU, and Z is A, C, or U. Although less is known about the linker region, ...

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Precise temporal control is needed for RNA viral genomes to translate sufficient replication-required products before clearing ribosomes and initiating replication. A 39 translational enhancer in Turnip crinkle virus (TCV) overlaps an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. The higher-order structure in the region was examined through alteration of critical sequences revealing novel interactions between an H-type pseudoknot and upstream residues, and between the TSS and internal and terminal loops of an upstream hairpin. Our results suggest that the TSS forms a stable scaffold that allows for simultaneous interactions with external sequences through base pairings on both sides of its large internal symmetrical loop. Binding of TCV RNA-dependent RNA polymerase (RdRp) to the region potentiates a widespread conformational shift with substantial rearrangement of the TSS region, including the element required for efficient ribosome binding. Degrading the RdRp caused the RNA to resume its original conformation, suggesting that the initial conformation is thermodynamically favored. These results suggest that the 39 end of TCV folds into a compact, highly interactive structure allowing RdRp access to multiple elements including the 39 end, which causes structural changes that potentiate the shift between translation and replication.

Ribosomal protein L3 (L3) is an essential and indispensable component for formation of the peptidyltransferase center. Atomic resolution ribosome structures reveal two extensions of L3 protruding deep into the core of the large subunit. The central extension of L3 in Saccharomyces cerevisiae was investigated using a combination of molecular genetic, biochemical, chemical probing, and molecular modeling methods. A reciprocal relationship between ribosomal affinity for eEF-1A stimulated binding of aa-tRNA and for eEF2 suggests that the central extension of L3 may function as an allosteric switch in coordinating binding of the elongation factors. Opening of the aa-tRNA accommodation corridor promoted resistance to the A site-specific translational inhibitor anisomycin, suggesting a competitive model for anisomycin resistance. These changes were also found to inhibit peptidyltransferase activity, stimulating programmed -1 ribosomal frameshifting and promoting virus propagation defects. These studies provide a basis for deeper insight into rational design of small molecule antiviral therapeutics.

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