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.
The genomes of positive-strand RNA viruses undergo conformational shifts that complicate efforts to equate structures with function. We have initiated a detailed analysis of secondary and tertiary elements within the 3 end of Turnip crinkle virus (TCV) that are required for viral accumulation in vivo. MPGAfold, a massively parallel genetic algorithm, suggested the presence of five hairpins (H4a, H4b, and previously identified hairpins H4, H5, and Pr) and one H-type pseudoknot (⌿ 3 ) within the 3-terminal 194 nucleotides (nt). In vivo compensatory mutagenesis analyses confirmed the existence of H4a, H4b, ⌿ 3 and a second pseudoknot (⌿ 2 ) previously identified in a TCV satellite RNA. In-line structure probing of the 194-nt fragment supported the coexistence of H4, H4a, H4b, ⌿ 3 and a pseudoknot that connects H5 and the 3 end (⌿ 1 ). Stepwise replacements of TCV elements with the comparable elements from Cardamine chlorotic fleck virus indicated that the complete 142-nt 3 end, and subsets containing ⌿ 3 , H4a, and H4b or ⌿ 3 , H4a, H4b, H5, and ⌿ 2 , form functional domains for virus accumulation in vivo. A new 3-D molecular modeling protocol (RNA2D3D) predicted that H4a, H4b, H5, ⌿ 3 , and ⌿ 2 are capable of simultaneous existence and bears some resemblance to a tRNA. The related Japanese iris necrotic ring virus does not have comparable domains. These results provide a framework for determining how interconnected elements participate in processes that require 3 untranslated region sequences such as translation and replication.Replication of plus-strand RNA viruses initially requires translation of the genomic RNA to produce the virus-encoded, RNA-dependent RNA polymerase (RdRp) and any auxiliary viral proteins necessary for transcription. In a process that is poorly defined but likely dictated by viral and/or cellular factors, translation is terminated and the genomic RNA becomes available for reiterative synthesis of complementary strands, a process that requires membrane association (1, 2, 26). For some viruses, subsequent viral plus-strand synthesis occurs in virus-specific membrane invaginations known as spherules, which contain a limited number of minus-sense genomes and whose formation is induced by specific viral proteins (1,15,26). Although the process of producing viral progeny has been extensively studied using many different viral systems, it remains poorly understood. For example, fundamental questions, such as the role that conformational shifts in RNA structure play in switching the template from translation to replication, the proteins required to enact such events, and if cis-acting core promoters, enhancers, and repressors are organized into functional, interacting modules, remain virtually unanswered.Recent reports that portions of RNA viral genomes undergo conformational shifts to execute different functions (10, 13, 21, 28) complicate efforts to assign biological roles to groups of cis-acting elements that may not structurally coexist. The ability of viral RNAs to assume multiple conformation...
Turnip crinkle virus (TCV) and its 356-nt satellite RNA satC share 151 nt of 3'-terminal sequence, which contain 8 positional differences and are predicted to fold into virtually identical structures, including a series of four phylogenetically inferred hairpins. SatC and TCV containing reciprocal exchanges of this region accumulate to only 15% or 1% of wild-type levels, respectively. Step-wise conversion of satC and TCV 3'-terminal sequences into the counterpart's sequence revealed the importance of having the cognate core promoter (Pr), which is composed of a single hairpin that differs in both sequence and stability, and an adjacent short 3'-terminal segment. The negative impact of the more stable TCV Pr on satC could not be attributed to lack of formation of a known tertiary interaction involving the 3'-terminal bases, nor an effect of coat protein, which binds specifically to TCV-like Pr and not the satC Pr. The satC Pr was a substantially better promoter than the TCV Pr when assayed in vitro using purified recombinant TCV RdRp, either in the context of satC or when assayed downstream of non-TCV-related sequence. Poor activity of the TCV Pr in vitro occurred despite solution structure probing indicating that its conformation in the context of satC is similar to the active form of the satC Pr, which is thought to form following a required conformational switch. These results suggest that evolution of satC following its initial formation generated a Pr that can function more efficiently in the absence of additional TCV sequence that may be required for full functionality of the TCV Pr.
Although a CCTG expansion in the gene encoding the zinc knuckle protein CNBP causes a common form of muscular dystrophy, the function of both human CNBP and its putative budding yeast ortholog Gis2 remain poorly understood. Here we report the protein interactions of Gis2 and the subcellular locations of both Gis2 and CNBP. We found that Gis2 exhibits RNA-dependent interactions with two proteins involved in mRNA recognition, the poly(A) binding protein and the translation initiation factor eIF4G. We show that Gis2 is a component of two large RNA-protein granules, processing bodies and stress granules, which contain translationally repressed mRNAs. Consistent with a functional ortholog, CNBP also associates with the poly(A) binding protein and accumulates in stress granules during arsenite treatment of human cells. These results implicate both Gis2 and CNBP in mRNA handling during stress.
The mutation frequency of Turnip crinkle virus can increase 12-fold without inducing error catastrophe. Lesions in a hairpin repressor frequently reverted and led to second-site alterations biased for specific mutations. These results suggest that the hairpin may also function as an RNA chaperone to properly fold the RNA-dependent RNA polymerase.Turnip crinkle virus (TCV), a member of the genus Carmovirus in the family Tombusviridae, has been used as a model for identifying cis-acting elements important for RNA replication. The 4,054-base TCV genome consists of a positive-sense, single-stranded RNA with five overlapping open reading frames (ORF) encoding proteins that function in replication, movement, and packaging (3,8,13) (Fig. 1A). p28 and its readthrough product p88 are required for replication in vivo. p88 contains the RNA-dependent RNA polymerase (RdRp) active site and can by itself promote complementary-strand synthesis from cognate templates in an in vitro (cell-free) assay (16).TCV is also associated with several noncoding satellite RNAs (satRNAs) that range from 194 to 356 bases, with satC (356 bases) sharing its 3Ј-terminal 166 bases with TCV (19). Based on studies using satC, several elements in the 3Ј untranslated region (3Ј UTR) of TCV that are likely important for replication have been identified. These include a core promoter hairpin (Pr) for synthesis of minus strands, which is located at the 3Ј terminus of plus strands and can function independently as a promoter in in vitro assays (2,20,22), and hairpin 5 (H5), a recently identified repressor of minus-strand synthesis (24) (Fig. 1B). Hairpins with similar sequence and/or structural features are located in analogous positions in nearly all carmoviruses (24). H5, which contains a large symmetrical internal loop (LSL), represses minus-strand synthesis in vitro when the 3Ј side of the LSL is base paired with the 3Ј-terminal 4 bases of the RNA (GCCC-OH), thereby sequestering the 3Ј terminus from the RdRp (Fig. 1B). Disruption of the interaction between the satC 3Ј terminus and the LSL substantially enhances synthesis of both full-length and aberrantly initiated complementary strands in vitro. In addition, localized disruption of H5 (and 3Ј-proximal sequences) occurred in satC transcripts containing a deletion of 3Ј-terminal bases. In TCV, compensatory exchanges between the LSL and 3Ј-terminal bases enhanced replication relative to that of virus containing the individual alterations, strongly suggesting that H5 serves an analogous function in TCV (unpublished data). A repressor of minus-strand synthesis with a large asymmetrical internal loop was also recently discovered in Tomato bushy stunt virus, a virus belonging to the same family as TCV (14). In the present study, we have identified an additional feature of H5. Mutations introduced into the TCV LSL resulted, surprisingly, in an increase of as much as 12-fold in second-site mutations scattered throughout the sequenced region; most of these alterations were uridylate-to-cytidylate or adenylate-...
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