Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA

Circle, D A; Lyons, Alita J.; Neel, Olivia D.; Robertson, Hugh D.

doi:10.1261/rna.2173903

Cited by 9 publications

(14 citation statements)

References 38 publications

(55 reference statements)

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“…UV cross-linking has been used for detecting tertiary interactions in vitro in a wide range of RNAs, for example, hepatitis delta virus (5,7,9), tRNAs (4), small nuclear RNAs (32), hairpin ribozymes (8), ribosomal RNAs (22), and Peach latent mosaic viroid (16). As shown here, this approach may be extended to investigate the in vivo existence of these tertiary interactions.…”

Section: Fig 3 Rna Gel Blotmentioning

confidence: 99%

Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo

Wang¹,

Zhong²,

Itaya³

et al. 2007

J Virol

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RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for RNA-RNA, RNA-protein, or RNA small ligand interactions. The structures of viral and viroid RNA motifs are studied commonly by in vitro, computational, and mutagenesis approaches. Demonstration of the in vivo existence of a motif will help establish its biological significance and promote mechanistic studies on its functions. By using UV cross-linking and primer extension, we have obtained direct evidence for the in vivo existence of the loop E motif of Potato spindle tuber viroid. We present our findings and discuss their biological implications.RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for interactions with proteins, other RNA motifs, or small ligands (19). For viral and viroid RNAs, such interactions are crucial for the establishment of infection. The structures of RNA motifs of viral and viroid RNAs have been generally studied by in vitro chemical/ enzymatic mapping, thermodynamic calculations using minimum free energy, biophysical characterization, and mutational analyses. It has been shown experimentally that some structural features of RNAs deduced by in vitro and in silico methods may not be identical to those found in vivo, likely because of the influence of cellular factors that interact with the RNAs (2, 26, 30). Therefore, to further establish the biological significance of findings from in vitro and in silico approaches, the in vivo existence of an RNA motif should be directly demonstrated.We are interested in using viroid infection as a model system to investigate the structure-function relationships of RNA motifs. Viroids are the smallest known nucleic acid-based infectious agents and self-replicating genetic units. Their genomes consist of single-stranded, circular RNAs ranging in size from 250 to 400 nucleotides (12, 13, 33). Viroids do not encode proteins, do not have encapsidation mechanisms, and do not require helper viruses. Nonetheless, they can replicate efficiently and spread throughout an infected plant (13, 33). Thus, viroid infection provides an excellent experimental system to investigate the basic RNA structure-function relationships for infection as well as for RNA biology (18).One of the best-studied viroid RNA motifs is the so-called loop E located in the central conserved region of Potato spindle tuber viroid (PSTVd). The loop E is a recurrent motif found in many RNAs, including 5S, 16S, and 23S rRNAs, group I and group II introns, bacterial RNase P, ribozyme of Tobacco ringspot virus satellite RNA (20), and lysine riboswitches (15,31), where it plays critical roles in RNA-RNA and RNA-protein interactions. UV-induced cross-linking between G98 and U260 in vitro provided the first evidence for the existence of local tertiary structure in the loop E of PSTVd resembling that of the loop E of 5S rRNA from HeLa cells (6) ...

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Section: Fig 3 Rna Gel Blotmentioning

confidence: 99%

Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo

Wang¹,

Zhong²,

Itaya³

et al. 2007

J Virol

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“…Affinity cleavage is a widely used technique to define structural features of complexes between proteins and nucleic acids (35)(36)(37). Application of the affinity cleaving technique is particularly appropriate in the study of PKR due to PKR's ability to bind many different RNAs with simple and complex structures (2,3,27,38,39). Furthermore, PKR's dsRBD is composed of two copies of the dsRBM and the precise role of the individual dsRBMs in PKR's binding mechanism is poorly understood.…”

Section: Affinity Cleavage Experiments Can Identify the Rna Binding S...mentioning

confidence: 99%

“…In vitro, PKR is activated by binding to RNA molecules with extensive duplex secondary structure (2). In vivo, the enzyme is believed to be activated by viral double-stranded RNA (dsRNA) or viral replicative intermediates comprising dsRNA (3). Activated PKR phosphorylates the alpha subunit of the heterotrimeric eukaryotic translation initiation factor 2 (eIF2R) (4).…”

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confidence: 99%

Identification of Binding Sites for Both dsRBMs of PKR on Kinase-Activating and Kinase-Inhibiting RNA Ligands

2002

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The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR has a double-stranded RNA-binding domain (dsRBD) composed of two copies of the dsRNA binding motif (dsRBM). PKR's dsRBD is involved in the regulation of the enzyme as dsRNAs of cellular and viral origins bind to the dsRBD, leading to either activation or inhibition of PKR's kinase activity. In this study, we site-specifically modified each of the dsRBMs of PKR's dsRBD with the hydroxyl radical generator EDTA small middle dotFe and performed cleavage studies on kinase-activating and kinase-inhibiting RNAs. These experiments led to the identification of binding sites for the individual dsRBMs on various RNA ligands including a viral activating RNA (TAR from HIV-1), a viral inhibiting RNA (VA(I) RNA from adenovirus), an aptamer RNA that activates PKR, and a small synthetic inhibiting RNA. These results indicate that some RNAs interact only with one dsRBM, while others can bind both dsRBMs of PKR. In addition, EDTA small middle dotFe modification coupled with site-directed mutagenesis was used to assess the extent of cooperativity in the binding of the two dsRBMs. These experiments support the hypothesis that simultaneous binding of both dsRBMs of PKR occurs on kinase activating RNA ligands.

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“…The HDV RNA genome is approximately 1700 nucleotides in length which encodes only one open reading frame. The genome has a highly conserved, noncoding ‘viroid‐like’ region that is vital for viral replication and a heterologous coding domain that determines two isoforms of HDV antigen , namely the small HDAg isoform of 195 amino acids (S‐HDAg) and the large HDAg isoform of 214 amino acids (L‐HDAg), which result from a single gene product by RNA editing .…”

Section: Introductionmentioning

confidence: 99%

Identification of a natural intergenotypic recombinant hepatitis delta virus genotype 1 and 2 in Vietnamese HBsAg‐positive patients

Nguyen

Toàn

et al. 2014

Journal of Viral Hepatitis

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Hepatitis D virus (HDV) infection is acquired as a co- /superinfection of Hepatitis B virus (HBV) and can modulate the pathophysiology of chronic hepatitis B and related liver diseases including hepatocellular carcinoma. Among the eight distinct HDV genotypes reported, relatively few studies have attempted to investigate the prevalence of HDV mixed genotypes and RNA recombination of HDV. With a recorded prevalence of 10-20% HBV infection in Vietnam, this study investigated the HDV variability, HDV genotypes and HDV recombination among twenty-one HDV isolates in Vietnamese HBsAg-positive patients. HDV subgenomic and full-length genome sequences were obtained using newly established HDV-specific RT-PCR techniques. The nucleotide homology was observed from 74.6% to 99.4% among the investigated full-length genome of the HDV isolates. We observed HDV genotype 1 and HDV genotype 2 in the investigated Vietnamese patients. Although no HDV genotype mixtures were observed, we report here a newly identified recombinant of HDV genotypes (HDV 1 and HDV 2). The identified recombinant HDV isolate C03 revealed sequence homology to both HDV genotype 1 (nt1 to nt907) and HDV genotype 2 (nt908 to nt1675; HDAg coding region) with a breakpoint at nt908. Our findings demonstrate the prevalence of intergenotypic recombination between HDV genotypes 1 and 2 in a Vietnamese HBsAg-positive patient. Extended investigation on the distribution and prevalence of HDV, HDV mixed genotypes and recombinant HDV genotypes in a larger Vietnamese population offers vital insights into understanding of the micro-epidemiology of HDV and subsequent pathophysiology in chronic HBV- /HDV-related liver diseases.

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Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA

Cited by 9 publications

References 38 publications

Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo

Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo

Identification of Binding Sites for Both dsRBMs of PKR on Kinase-Activating and Kinase-Inhibiting RNA Ligands

Identification of a natural intergenotypic recombinant hepatitis delta virus genotype 1 and 2 in Vietnamese HBsAg‐positive patients

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