Protein synthesis of an RNA template can initiate by two different known mechanisms: capdependent translation initiation and cap-independent translation initiation. The latter is driven by RNA sequences called internal ribosome entry sites (IRESs) that are found in both viral RNAs and cellular mRNAs. The diverse mechanisms used by IRESs are reflected in their structural diversity, and this structural diversity challenges us to develop a cohesive model linking IRES function to structure. With more direct structural information available for the viral IRESs, data suggest an inverse correlation between the degree to which an IRES RNA can form a stable structure on its own, and the number of factors that it requires to function. Lessons learned from the viral IRESs may help understand the cellular IRESs, although more structural data is needed before any strong links can be made.

RNA viruses co-opt the host cell's biological machinery, and their infection strategies often depend on specific structures in the viral genomic RNA. Examples are tRNA-like structures (TLSs), found at the 39 end of certain plant viral RNAs, which can use the cell's aminoacyl tRNA-synthetases (AARSs) to drive addition of an amino acid to the 39 end of the viral RNA. TLSs are multifunctional RNAs involved in processes such as viral replication, translation, and viral RNA stability; these functions depend on their fold. Experimental result-based structural models of TLSs have been published. In this study, we further examine these structures using a combination of biophysical and biochemical approaches to explore the three-dimensional (3D) architectures of TLSs from the turnip yellow mosaic virus (TYMV), tobacco mosaic virus (TMV), and brome mosaic virus (BMV). We find that despite similar function, these RNAs are biophysically diverse: the TYMV TLS adopts a characteristic tRNA-like L shape, the BMV TLS has a large compact globular domain with several helical extensions, and the TMV TLS aggregates in solution. Both the TYMV and BMV TLS RNAs adopt structures with tight backbone packing and also with dynamic structural elements, suggesting complexities and subtleties that cannot be explained by simple tRNA mimicry. These results confirm some aspects of existing models and also indicate how these models can be improved. The biophysical characteristics of these TLSs show how these multifunctional RNAs might regulate various viral processes, including negative strand synthesis, and also allow comparison with other structured RNAs.

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 d 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.

The hepatitis C virus (HCV) internal ribosome entry site (IRES) drives non-canonical initiation of protein synthesis necessary for viral replication. HCV IRES functional studies have focused on 80S ribosome formation, but have not explored roles after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit’s decoding groove and cause only a local perturbation in IRES structure result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, we find the mutation decreases IRES activity by inhibiting the first ribosome translocation event, and modeling suggests that this effect is through an interaction with a single ribosomal protein. The HCV IRES’ ability to manipulate the ribosome provides insight into how the ribosome’s structure and function can be altered by bound RNAs, including those derived from cellular invaders.

Cap-dependent translation initiation in eukaryotes is a complex process involving many factors and serves as the primary mechanism for eukaryotic translation (37, 44). The first step in the initiation process, recruitment of the m 7 G (7-methylguanosine)-capped mRNA to the ribosome, is widely considered the rate-limiting step. It begins with recognition of and binding to the m 7 G cap at the 5Ј end of the mRNA by the eukaryotic translation initiation factor 4F (eIF4F) complex, which contains three proteins: eIF4E (a cap-binding protein), eIF4G (a scaffold protein with RNA binding sites), and eIF4A (an RNA helicase). eIF4G's interaction with eIF3, itself a multisubunit complex that interacts with the 40S ribosome, facilitates the actual recruitment of capped RNA to the ribosome. With the help of several other initiation factors, the small ribosomal subunit scans the mRNA from 5Ј to 3Ј until a translation initiation codon (AUG) in appropriate context is identified and an 80S ribosomal complex is formed, after which the first peptide bond is formed, thus ending the initiation process (37, 44). The AUG context can play an important role in the efficiency of translation initiation (23, 44). The length, structure, and presence of AUGs or open reading frames in the mRNA 5Ј untranslated region (UTR) can negatively affect cap-dependent translation and ribosomal scanning. In general, long and highly structured 5Ј UTRs, as well as upstream AUGs leading to short open reading frames, can impede ribosome scanning and lead to reduced translation (23, 44). In addition, 5Ј UTRs less than 10 nucleotides (nt) in length are thought to be too short to enable preinitiation complex assembly and scanning (24). Thus, several attributes of the mRNA 5Ј UTR are known to negatively affect translation initiation, whereas only the AUG context and the absence of negative elements are known to have a positive effect on translation initiation (44).Two of the important mRNA features associated with capdependent translation, the cap and the 5Ј UTR, are significantly altered by an RNA processing event known as spliced leader (SL) trans splicing (3,8,17,26,36,47). This takes place in members of a diverse group of eukaryotic organisms, including some protozoa, sponges, cnidarians, chaetognaths, flatworms, nematodes, rotifers, crustaceans, and tunicates (17,28,39,55,56). In SL trans splicing, a separately transcribed small exon (16 to 51 nucleotides [nt]) with its own cap gets added to the 5Ј end of pre-mRNAs. This produces mature mRNAs with a unique cap and a conserved sequence in the 5Ј UTR. In metazoa, the m 7 G cap is replaced with a trimethylguanosine (TMG) cap (m 2,2,7 GpppN) (27,30,46,49). In nematodes, ϳ70% of all mRNAs are trans spliced and therefore have a TMG cap and an SL (2). In general, eukaryotic eIF4E proteins do not effectively recognize the TMG cap (35). This raises the issues of how the translation machinery in trans-splicing