Cell Biology & Molecular Genetics
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Item TRANSLATION AND MOVEMENT OF AN INFECTIOUS UMBRAVIRUS-LIKE RNA CITRUS YELLOW VEIN ASSOCIATED VIRUS(2021) Liu, Jingyuan; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While the citrus yellow vein disease was first reported more than 60 years ago, its causal agent, citrus yellow vein associated virus (CYVaV), was only sequenced in 2013. CYVaV was recently structurally characterized and phylogenetically classified as a Class 2 umbravirus-like associated RNA (ulaRNA), a new category of coat-protein dependent subviral RNA replicons. There is a dearth of structural and biological characterizations of ulaRNAs as well as studies on their translation regulation. CYVaV has a limited genome size (2.7 kb), and contains only two ORFs that encode replicase proteins p21 and p81. Here I show that CYVaV transcripts are infectious in Arabidopsis protoplasts and Nicotiana benthamiana plants, and CYVaV can systemically infect the latter using agro-infiltration, despite the absence of encoded movement proteins or silencing suppressors. Fluorescent in situ hybridization (FISH) revealed that CYVaV is phloem-limited, and restricted to sieve elements, companion cells, and phloem parenchyma cells. In this work, the secondary structures of the CYVaV 5ʹ end and 3ʹ UTR were determined using SHAPE structure probing and phylogenic comparisons, and were used to infer the putative structures of other Class 2 ulaRNAs, revealing a number of distinctive structural features. Here I report the identification of a novel 3ʹCITE in the 3ʹUTR of CYVaV that is strongly conserved in Class 2 ulaRNAs and structurally resembles an I-shaped structure (ISS) 3ʹCITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5ʹ end. We also report that the ~30 nt 5ʹ terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3ʹCITE, or the 3ʹCITEs of umbravirus PEMV2, or even independent of a 3ʹCITE. These findings introduce a new type of 3ʹCITE and provide the first information on translation of ulaRNAs.Item Alternate Conformations Regulate Ribosomal Recoding in a Positive-sense RNA Virus(2016) Kuhlmann, Micki Michelle; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Positive-sense RNA viruses are important animal, plant, insect and bacteria pathogens and constitute the largest group of RNA viruses. Due to the relatively small size of their genomes, these viruses have evolved a variety of non-canonical translation mechanisms to optimize coding capacity expanding their proteome diversity. One such strategy is codon redefinition or recoding. First described in viruses, recoding is a programmed translation event in which codon alterations are context dependent. Recoding takes place in a subset of messenger RNA (mRNAs) with some products reflecting new, and some reflecting standard, meanings. The ratio between the two is both critical and highly regulated. While a variety of recoding mechanisms have been documented, (ribosome shunting, stop-carry on, termination-reinitiation, and translational bypassing), the two most extensively employed by RNA viruses are Programmed Ribosomal Frameshifting (PRF) and Programmed Ribosomal Readthrough (PRT). While both PRT and PRF subvert normal decoding for expression of C-terminal extension products, the former involves an alteration of reading frame, and the latter requires decoding of a non-sense codon. Both processes occur at a low but defined frequency, and both require Recoding Stimulatory Elements (RSE) for regulation and optimum functionality. These stimulatory signals can be embedded in the RNA in the form of sequence or secondary structure, or trans-acting factors outside the mRNA such as proteins or micro RNAs (miRNA). Despite 40+ years of study, the precise mechanisms by which viral RSE mediate ribosome recoding for the synthesis of their proteins, or how the ratio of these products is maintained, is poorly defined. This study reveals that in addition to a long distance RNA:RNA interaction, three alternate conformations and a phylogenetically conserved pseudoknot regulate PRT in the carmovirus Turnip crinkle virus (TCV).Item STRUCTURAL ANALYSIS OF RIBOSOME BINDING ELEMENTS OF THE 3´ UTR IN TWO POSITIVE-STRAND PLANT RNA VIRUSES(2016) Le, My Tra; Simon, Anne E; Dayie, Kwaku T; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Turnip crinkle virus (TCV) and Pea enation mosaic virus (PEMV) are two positive (+)-strand RNA viruses that are used to investigate the regulation of translation and replication due to their small size and simple genomes. Both viruses contain cap-independent translation elements (CITEs) within their 3´ untranslated regions (UTRs) that fold into tRNA-shaped structures (TSS) according to nuclear magnetic resonance and small angle x-ray scattering analysis (TCV) and computational prediction (PEMV). Specifically, the TCV TSS can directly associate with ribosomes and participates in RNA-dependent RNA polymerase (RdRp) binding. The PEMV kissing-loop TSS (kl-TSS) can simultaneously bind to ribosomes and associate with the 5´ UTR of the viral genome. Mutational analysis and chemical structure probing methods provide great insight into the function and secondary structure of the two 3´ CITEs. However, lack of 3-D structural information has limited our understanding of their functional dynamics. Here, I report the folding dynamics for the TCV TSS using optical tweezers (OT), a single molecule technique. My study of the unfolding/folding pathways for the TCV TSS has provided an unexpected unfolding pathway, confirmed the presence of Ψ3 and hairpin elements, and suggested an interconnection between the hairpins and pseudoknots. In addition, this study has demonstrated the importance of the adjacent upstream adenylate-rich sequence for the formation of H4a/Ψ3 along with the contribution of magnesium to the stability of the TCV TSS. In my second project, I report on the structural analysis of the PEMV kl-TSS using NMR and SAXS. This study has re-confirmed the base-pair pattern for the PEMV kl-TSS and the proposed interaction of the PEMV kl-TSS with its interacting partner, hairpin 5H2. The molecular envelope of the kl-TSS built from SAXS analysis suggests the kl-TSS has two functional conformations, one of which has a different shape from the previously predicted tRNA-shaped form. Along with applying biophysical methods to study the structural folding dynamics of RNAs, I have also developed a technique that improves the production of large quantities of recombinant RNAs in vivo for NMR study. In this project, I report using the wild-type and mutant E.coli strains to produce cost-effective, site-specific labeled, recombinant RNAs. This technique was validated with four representative RNAs of different sizes and complexity to produce milligram amounts of RNAs. The benefit of using site-specific labeled RNAs made from E.coli was demonstrated with several NMR techniques.Item DIFFERENT REPLICATION REQUIREMENTS IN THE HOMOLOGOUS 3' ENDS OF A POSITIVE STRAND RNA VIRUS AND ITS SUBVIRAL RNA(2010) Guo, Rong; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)SatC is a noncoding subviral RNA associated with Turnip Crinkle Virus (TCV), a small (4054 nt) single-stranded (+)-strand RNA virus belonging to the Carmovirus genus. Because of its small size (356 nt) and TCV-derived 3' end, satC has been successfully used as a model to elucidate sequence and structural requirements for TCV RNA replication. Although satC is considered a model to identify cis-acting elements required for TCV replication, recent findings indicate distinct differences in structures and functions of these related sequences. RNA2D3D predicts that part of the TCV 3' end (H5, H4a, H4b and two pseudoknots) folds into an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits and is required for translation. SatC contains a similar 3' end with 6 nt differences in the 100 nt TSS region. RNA2D3D did not predict a similar structure for satC TSS region, and satC did not bind yeast ribosomes. satC nucleotides were changed into TCV TSS bases to determine which base differences are responsible for the loss of the TSS in satC. Changing these bases all increased ribosome binding but surprisingly none of them had an effect on satC accumulation in protoplasts and plants. Therefore satC may need these and other 3' end base differences for its required conformational switch for efficient replication, and not to inhibit ribosome binding. In vivo genetic selection (SELEX) of the linker sequence between H5 and the Pr showed the conservation of UCC, which led to the discovery of Ø2. Ø2 is required for both viral and satC accumulation in protoplasts. H5-Pr linker had no significant structural change after RdRp binding in satC, which is different with TCV H5-Pr linker. TCV H5-Pr linker had a major structural change upon RdRp binding, and is proposed to be involved in a conformational switch. Replacement of satC H4a with randomized sequence and scoring for fitness in plants by SELEX resulted in winning sequences that contain an H4a-like stem-loop. SELEX of H4a/H4b in satC generated two different structures: wt H4a/H4b-like structure and a single hairpin structure. Two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts. With the protection of CP, satC can form higher amount of dimers that have additional nucleotides at the junction sites in the absence of TCV. The extra nucleotides are not necessarily associated with an active TCV RdRp.Item Cis-acting elements and a conformational switch involved in replication of a positive-strand RNA virus(2006-05-04) Zhang, Jiuchun; Simon, Anne E.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Replication of positive (+)-strand RNA virus genomes is a fundamental process in a virus's life cycle. Turnip crinkle virus (TCV) and an associated satellite RNA (satC) share 151 nt of 3' terminal sequence, which are predicted to fold into four phylogenetically-inferred hairpins (from the 3' end: Pr hairpin, H5, H4b and H4a). Pr hairpin is part of the core promoter (Pr) required for satC (Song and Simon, 1995) and TCV (Sun and Simon, in press) negative (-)-strand synthesis. To identify other regulatory cis-acting elements throughout satC, individual deletions of six other predicted hairpins (H5, H4b, H4a, M1H, H6, and H2) were performed. These deletions significantly reduce accumulation of (+)-strand monomers and differentially affect accumulation of (+)-strand dimers and (-)-strands in Arabidopsis protoplasts. Results from in vivo genetic selection and mutational analyses of satC H5 indicate that robust satC accumulation in vivo requires specific sequences in the large symmetrical internal loop (LSL) and a stable stem and specific base pairs in the lower stem. The upper stem-loop has considerable plasticity. Moreover, H5 may be involved in accumulation of both strands. Mutational analyses also suggest that the LSL and/or the 3' terminus may have other functions in addition to forming a pseudoknot (pseudoknot 1), which is required for replication. An RNA conformational switch from a pre-active structure to an active structure appears required to regulate initiation of satC (-)-strand synthesis (Zhang et al., 2006). Results from mutational analyses suggest that H4a and H4b function as a unit and pseudoknot 2, formed between H4b and sequence flanking the 3' side of H5 and whose disruption reduces satC accumulation in vivo, stabilizes the pre-active satC structure. In addition, an upstream element (DR) may help to promote the switch. Step-wise conversion of satC and TCV 3' terminal homologous sequences into the counterpart's sequence revealed the importance of having the cognate core promoter. The satC Pr is a substantially better promoter than the TCV Pr when assayed in vitro. These results suggest that the TCV Pr requires upstream elements for full functionality and that evolution of satC generated a Pr that functions more efficiently by itself.