Translation-Independent Roles of RNA Secondary Structures within the Replication Protein Coding Region of Turnip Crinkle Virus

RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.

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Influence of the Leader protein coding region of foot-and-mouth disease virus on virus replication

Journal of General Virology ◽

10.1099/vir.0.052126-0 ◽

2013 ◽

Vol 94 (7) ◽

pp. 1486-1495 ◽

Cited By ~ 17

Author(s):

Graham J. Belsham

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Initiation Codon ◽

Coding Region ◽

Protein Coding ◽

Coding Sequence ◽

Bhk Cells ◽

Leader Protein ◽

Mouth Disease Virus

The foot-and-mouth disease virus (FMDV) Leader (L) protein is produced in two forms, Lab and Lb, differing only at their amino-termini, due to the use of separate initiation codons, usually 84 nt apart. It has been shown previously, and confirmed here, that precise deletion of the Lab coding sequence is lethal for the virus, whereas loss of the Lb coding sequence results in a virus that is viable in BHK cells. In addition, it is now shown that deletion of the ‘spacer’ region between these two initiation codons can be tolerated. Growth of the virus precisely lacking just the Lb coding sequence resulted in a previously undetected accumulation of frameshift mutations within the ‘spacer’ region. These mutations block the inappropriate fusion of amino acid sequences to the amino-terminus of the capsid protein precursor. Modification, by site-directed mutagenesis, of the Lab initiation codon, in the context of the virus lacking the Lb coding region, was also tolerated by the virus within BHK cells. However, precise loss of the Lb coding sequence alone blocked FMDV replication in primary bovine thyroid cells. Thus, the requirement for the Leader protein coding sequences is highly dependent on the nature and extent of the residual Leader protein sequences and on the host cell system used. FMDVs precisely lacking Lb and with the Lab initiation codon modified may represent safer seed viruses for vaccine production.

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Protein-coding structured RNAs: A computational survey of conserved RNA secondary structures overlapping coding regions in drosophilids

Biochimie ◽

10.1016/j.biochi.2011.07.023 ◽

2011 ◽

Vol 93 (11) ◽

pp. 2019-2023 ◽

Cited By ~ 8

Author(s):

Sven Findeiß ◽

Jan Engelhardt ◽

Sonja J. Prohaska ◽

Peter F. Stadler

Keyword(s):

Secondary Structures ◽

Protein Coding ◽

Rna Secondary Structures ◽

Coding Regions

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The Infectivities of Turnip Yellow Mosaic Virus Genomes with Altered tRNA Mimicry Are Not Dependent on Compensating Mutations in the Viral Replication Protein

Journal of Virology ◽

10.1128/jvi.74.18.8368-8375.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8368-8375 ◽

Cited By ~ 5

Author(s):

Sergei A. Filichkin ◽

Kay L. Bransom ◽

Joel B. Goodwin ◽

Theo W. Dreher

Keyword(s):

Mosaic Virus ◽

Replication Protein ◽

Yellow Mosaic Virus ◽

Turnip Yellow Mosaic Virus ◽

Wild Type ◽

Coding Region ◽

Protein Coding ◽

Complete Sequencing ◽

Strand Initiation ◽

Virus Genomes

ABSTRACT Five highly infectious turnip yellow mosaic virus (TYMV) genomes with sequence changes in their 3′-terminal regions that result in altered aminoacylation and eEF1A binding have been studied. These genomes were derived from cloned parental RNAs of low infectivity by sequential passaging in plants. Three of these genomes that are incapable of aminoacylation have been reported previously (J. B. Goodwin, J. M. Skuzeski, and T. W. Dreher, Virology 230:113–124, 1997). We now demonstrate by subcloning the 3′ untranslated regions into wild-type TYMV RNA that the high infectivities and replication rates of these genomes compared to their progenitors are mostly due to a small number of mutations acquired in the 3′ tRNA-like structure during passaging. Mutations in other parts of the genome, including the replication protein coding region, are not required for high infectivity but probably do play a role in optimizing viral amplification and spread in plants. Two other TYMV RNA variants of suboptimal infectivities, one that accepts methionine instead of the usual valine and one that interacts less tightly with eEF1A, were sequentially passaged to produce highly infectious genomes. The improved infectivities of these RNAs were not associated with increased replication in protoplasts, and no mutations were acquired in their 3′ tRNA-like structures. Complete sequencing of one genome identified two mutations that result in amino acid changes in the movement protein gene, suggesting that improved infectivity may be a function of improved viral dissemination in plants. Our results show that the wild-type TYMV replication proteins are able to amplify genomes with 3′ termini of variable sequence and tRNA mimicry. These and previous results have led to a model in which the binding of eEF1A to the 3′ end to antagonize minus-strand initiation is a major role of the tRNA-like structure.

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Structure of the human lactate dehydrogenase B gene

Biochemical Journal ◽

10.1042/bj2570921 ◽

1989 ◽

Vol 257 (3) ◽

pp. 921-924 ◽

Cited By ~ 28

Author(s):

T Takeno ◽

S S L Li

Keyword(s):

Lactate Dehydrogenase ◽

Translation Initiation ◽

Initiation Site ◽

Initiation Codon ◽

Translation Initiation Site ◽

Coding Region ◽

Protein Coding ◽

Coding Sequence ◽

Human Genomic ◽

Human And Mouse

Human genomic clones containing parts of the lactate dehydrogenase B (LDH-B) gene (approx. 25 kb in length) were isolated and characterized. The protein-coding sequence of human LDH-B gene is interrupted by six introns at codons nos. 42-43, 82, 140, 198, 237 and 278-279, and the positions of these introns are homologous to those of LDH-A genes from man and mouse. The 5' non-coding region of human LDH-B gene is interrupted by an intron six nucleotide residues upstream of the ATG translation-initiation site, whereas those of human and mouse LDH-A genes are interrupted at 24 nucleotide residues 5' to the ATG initiation codon. As is the case of LDH-A genes from man and mouse, there is no intron in the 3' non-coding region of human LDH-B gene.

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The complete mitochondrial genome of Orancistrocerus aterrimus aterrimus and comparative analysis in the family Vespidae (Hymenoptera, Vespidae, Eumeninae)

ZooKeys ◽

10.3897/zookeys.790.25356 ◽

2018 ◽

Vol 790 ◽

pp. 127-144 ◽

Cited By ~ 2

Author(s):

Qiao-Hua Zhang ◽

Pan Huang ◽

Bin Chen ◽

Ting-Jing Li

Keyword(s):

Mitochondrial Genome ◽

Stop Codon ◽

Translation Termination ◽

Complete Mitochondrial Genome ◽

Rrna Genes ◽

Trna Genes ◽

Coding Region ◽

Protein Coding ◽

Tandem Repeat Sequences ◽

The Family

To date, only one mitochondrial genome (mitogenome) in the Eumeninae has been reported in the world and this is the first report in China. The mitogenome ofO.a.aterrimusis 17 972 bp long, and contains 38 genes, including 13 protein coding genes (PCGs), 23 tRNA genes, two rRNA genes, a long non-coding region (NCR), and a control region (CR). The mitogenome has 79.43% A + T content, its 13 PCGs use ATN as the initiation codon except forcox1using TTG, and nine genes used complete translation termination TAA and four genes have incomplete stop codon T (cox2,cox3,nad4, andcytb). Twenty-two of 23 tRNAs can form the typical cloverleaf secondary structure except fortrnS1. The CR is 1 078 bp long with 84.69% A+T content, comprising 28 bp tandem repeat sequences and 13 bp T-strech. There are two gene rearrangements which are an extratrnM2located betweentrnQandnad2and thetrnL2in the upstream ofnad1. Within all rearrangements of these mitogenomes reported in the family Vespidae, the translocation betweentrnS1andtrnEgenes only appears in Vespinae, and the translocation oftrnYin Polistinae and Vespinae. The absent codons of 13 PCGs in Polistinae are more than those both in Vespinae and Eumeninae in the family Vespidae. The study reports the complete mitogenome ofO.a.aterrimus, compares the characteristics and construct phylogenetic relationships of the mitogenomes in the family Vespidae.

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Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

BioMed Research International ◽

10.1155/2013/637850 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5 ◽

Cited By ~ 2

Author(s):

Ching-Fang Lai ◽

Chih-Ying Chen ◽

Lo-Chun Au

Keyword(s):

Gene Silencing ◽

Thermodynamic Stability ◽

Untranslated Region ◽

Stop Codon ◽

Secondary Structures ◽

Transcriptional Gene Silencing ◽

Small Interfering Rnas ◽

Coding Region ◽

Post Transcriptional Gene Silencing ◽

Flanking Sequences

Small interfering RNAs (siRNAs) are applied for post-transcriptional gene silencing by binding target mRNA. A target coding region is usually chosen, although the3′-untranslated region (3′-UTR) can also be a target. This study elucidates whether the coding region or3′-UTR elicits higher repression. pFLuc and pRLuc are two reporter plasmids. A segment ofFLucgene was PCR-amplified and inserted behind the stop codon of theRLucgene of the pRLuc. Similarly, a segment ofRLucgene was inserted behind the stop codon ofFLuc. Two siFLuc and two siRLuc were siRNAs designed to target the central portions of these segments. Therefore, the siRNA encountered the same targets and flanking sequences. Results showed that the two siFLuc elicited higher repression when theFLucsegment resided in the coding region. Conversely, the two siRLuc showed higher repression when theRLucsegment was in the3′-UTR. These results indicate that both the coding region and the3′-UTR can be more effective targets. The thermodynamic stability of the secondary structures was analyzed. The siRNA elicited higher repression in the coding region when the target configuration was stable, and needed to be solved by translation. A siRNA may otherwise favor the target at3′-UTR.

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RNA In Silico The Computational Biology of RNA Secondary Structures

Advances in Complex Systems ◽

10.1142/s0219525999000059 ◽

1999 ◽

Vol 02 (01) ◽

pp. 65-90 ◽

Cited By ~ 19

Author(s):

Chirstoph Flamm ◽

Ivo L. Hofacker ◽

Peter F. Stadler

Keyword(s):

Secondary Structure ◽

Fitness Landscape ◽

Rna Virus ◽

Point Mutations ◽

Secondary Structures ◽

Hill Climbing ◽

Stable States ◽

Punctuated Equilibria ◽

Rna Secondary Structures

RNA secondary structures provide a unique computer model for investigating the most important aspects of structural and evolutionary biology. The existence of efficient algorithms for solving the folding problem, i.e., for predicting the secondary structure given only the sequence, allows the construction of realistic computer simulations. The notion of a "landscape" underlies both the structure formation (folding) and the (in vitro) evolution of RNA. Evolutionary adaptation may be seen as hill climbing process on a fitness landscape which is determined by the phenotype of the RNA molecule (within the model this is its secondary structure) and the selection constraints acting on the molecules. We find that a substantial fraction of point mutations do not change an RNA secondary structure. On the other hand, a comparable fraction of mutations leads to very different structures. This interplay of smoothness and ruggedness (or robustness and sensitivity) is a generic feature of both RNA and protein sequence-structure maps. Its consequences, "shape space covering" and "neutral networks" are inherited by the fitness landscapes and determine the dynamics of RNA evolution. Punctuated equilibria at phenotype level and a diffusion like evolution of the underlying genotypes are a characteristics feature of such models. As a practical application of these theoretical findings we have designed an algorithm that finds conserved (and therefore potentially functional substructures of RNA virus genomes from spares data sets. The folding dynamics of particular RNA molecule can also be studied successfully based on secondary structure. Given an RNA sequence, we consider the energy landscape formed by all possible conformations (secondary structures). A straight formward implementation of the Metropolis algorithm is sufficient to produce a quite realistic folding kinetics, allowing to identify meta-stable states and folding pathways. Just as in the protein case there are good and bad folders which can be distinguished by the properties of their landscapes.

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CASQ1 Gene Is an Unlikely Candidate for Malignant Hyperthermia Susceptibility in the North American Population

Anesthesiology ◽

10.1097/01.anes.0000530185.78660.da ◽

2013 ◽

Vol 118 (2) ◽

pp. 344-349 ◽

Cited By ~ 24

Author(s):

Natalia Kraeva ◽

Elena Zvaritch ◽

Wanda Frodis ◽

Olga Sizova ◽

Alexander Kraev ◽

...

Keyword(s):

Malignant Hyperthermia ◽

North American ◽

Malignant Hyperthermia Susceptibility ◽

North American Population ◽

American Population ◽

Coding Region ◽

Protein Coding ◽

Coding Sequence ◽

Protein Levels ◽

The North

Abstract Background Malignant hyperthermia (MH, MIM# 145600) is a complex pharmacogenetic disorder that is manifested in predisposed individuals as a potentially lethal reaction to volatile anesthetics and depolarizing muscle relaxants. Studies of CASQ1-null mice have shown that CASQ1, encoding calsequestrin 1, the major Ca2+ binding protein in the lumen of the sarcoplasmic reticulum, is a candidate gene for MH in mice. The aim of this study was to establish whether the CASQ1 gene is associated with MH in the North American population. Methods The entire coding region of CASQ1 in 75 unrelated patients diagnosed by caffeine-halothane contracture test as MH susceptible (MHS) was analyzed by DNA sequencing. Subsequently, three groups of unrelated individuals (130 MHS, 100 MH negative, and 192 normal controls) were genotyped for a variant that was identified by sequencing. Levels of CASQ1 expression in the muscle from unrelated MHS and MH negative individuals were estimated by Western blotting. Results Screening of the entire coding sequence of the CASQ1 gene in 75 MHS patients revealed a single variant c.260T > C (p.Met87Thr) in exon 1. This variant is unlikely to be pathogenic, because its allele frequency in the MHS group was not significantly different from that of controls. There was also no difference in calsequestrin 1 protein levels between muscle samples from MHS and controls, including those carrying the p.Met87Thr variant. Conclusions This study revealed a low level of protein coding sequence variability within the human CASQ1 gene, indicating that CASQ1 is not a major MHS locus in the North American population.

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