scholarly journals RNA-Based Regulation of Transcription and Translation of Aureusvirus Subgenomic mRNA1

2009 ◽  
Vol 83 (19) ◽  
pp. 10096-10105 ◽  
Author(s):  
Wei Xu ◽  
K. Andrew White
Keyword(s):  
Coat Protein ◽  
Mutational Analysis ◽  
Regulation Of Transcription ◽  
Rna Structures ◽  
Rna Sequences ◽  
Stem Loop ◽  
Translational Enhancer ◽  
Stem Loop Structure ◽  
Regulatory Strategies ◽  
Rna Interaction

ABSTRACT Cucumber leaf spot virus (CLSV) is an aureusvirus (family Tombusviridae) that has a positive-sense RNA genome encoding five proteins. During infections, CLSV transcribes two subgenomic (sg) mRNAs and the larger of the two, sg mRNA1, encodes coat protein. Here, the viral RNA sequences and structures that regulate transcription and translation of CLSV sg mRNA1 were investigated. A medium-range RNA-RNA interaction in the CLSV genome, spanning 148 nucleotides, was found to be required for the efficient transcription of sg mRNA1. Further analysis indicated that the structure formed by this interaction acted as an attenuation signal required for transcription of sg mRNA1 via a premature termination mechanism. Translation of coat protein from sg mRNA1 was determined to be facilitated by a 5′-terminal stem-loop structure in the message that resembled a tRNA anticodon stem-loop. The results from mutational analysis indicated that the 5′-terminal stem-loop mediated efficient base pairing with a 3′-cap-independent translational enhancer at the 3′ end of the message, leading to efficient translation of coat protein from sg mRNA1. Comparison of the regulatory RNA structures for sg mRNA1 of CLSV to those used by the closely related tombusviruses and certain cellular RNAs revealed interesting differences and similarities that provide evolutionary and mechanistic insights into RNA-based regulatory strategies.

scholarly journals Ribosomal pausing during translation of an RNA pseudoknot.

1993 ◽  
Vol 13 (11) ◽  
pp. 6931-6940 ◽  
Author(s):  
P Somogyi ◽  
A J Jenner ◽  
I Brierley ◽  
S C Inglis
Keyword(s):  
High Efficiency ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Stem Loop ◽  
Pseudoknot Structure ◽  
Stem Loop Structure ◽  
Dead End ◽  
Rna Pseudoknot ◽  
Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

scholarly journals Novel Computational Method to Define RNA PSRs Explains Influenza A Virus Nucleotide Conservation

2018 ◽  
Author(s):  
Andrey Chursov ◽  
Nathan Fridlyand ◽  
Albert A. Sufianov ◽  
Oleg I. Kiselev ◽  
Irina Baranovskaya ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
H1n1 Influenza ◽  
Computational Method ◽  
Structural Elements ◽  
Rna Structures ◽  
Stem Loop ◽  
Rna Molecules ◽  
Stem Loop Structure ◽  
Evolutionarily Conserved

ABSTRACTRNA molecules often fold into evolutionarily selected functional structures. Yet, the literature offers neither a satisfactory definition for “structured RNA regions”, nor a computational method to accurately identify such regions. Here, we define structured RNA regions based on the premise that both stems and loops in functional RNA structures should be conserved among RNA molecules sharing high sequence homology. In addition, we present a computational approach to identify RNA regions possessing evolutionarily conserved secondary structures, RNA ISRAEU (RNA Identification of Structured Regions As Evolutionary Unchanged). Applying this method to H1N1 influenza mRNAs revealed previously unknown structured RNA regions that are potentially essential for viral replication and/or propagation. Evolutionary conservation of RNA structural elements may explain, in part, why mutations in some nucleotide positions within influenza mRNAs occur significantly more often than in others. We found that mutations occurring in conserved nucleotide positions may be more disruptive for structured RNA regions than single nucleotide polymorphisms in positions that are more prone to changes. Finally, we predicted computationally a previously unknown stem-loop structure and demonstrated that oligonucleotides complementing the stem (but not the loop or unrelated sequences) reduce viral replicationin vitro.These results contribute to understanding influenza A virus evolution and can be applied to rational design of attenuated vaccines and/or drug designs based on disrupting conserved RNA structural elements.AUTHOR SUMMARYRNA structures play key biological roles. However, the literature offers neither a satisfactory definition for “structured RNA regions” nor the computational methodology to identify such regions. We define structured RNA regions based on the premise that functionally relevant RNA structures should be evolutionarily conserved, and devise a computational method to identify RNA regions possessing evolutionarily conserved secondary structural elements. Applying this method to influenza virus mRNAs of pandemic and seasonal H1N1 influenza A virus generated Predicted Structured Regions (PSRs), which were previously unknown. This explains the previously mysterious sequence conservation among evolving influenza strains. Also, we have experimentally supported existence of a computationally predicted stem-loop structure predicted computationally. Our approach may be useful in designing live attenuated influenza vaccines and/or anti-viral drugs based on disrupting necessary conserved RNA structures.

scholarly journals mRNA Secondary Structure Modulates Translation of Tat-Dependent Formate Dehydrogenase N

2004 ◽  
Vol 186 (18) ◽  
pp. 6311-6315 ◽  
Author(s):  
Claire Punginelli ◽  
Bérengère Ize ◽  
Nicola R. Stanley ◽  
Valley Stewart ◽  
Gary Sawers ◽  
...  
Keyword(s):  
Formate Dehydrogenase ◽  
Mutational Analysis ◽  
Signal Sequence ◽  
Fold Increase ◽  
Bioinformatic Analysis ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Dependent Signal ◽  
Membrane Bound

ABSTRACT Formate dehydrogenase N (FDH-N) of Escherichia coli is a membrane-bound enzyme comprising FdnG, FdnH, and FdnI subunits organized in an (αβγ)3 configuration. The FdnG subunit carries a Tat-dependent signal peptide, which localizes the protein complex to the periplasmic side of the membrane. We noted that substitution of the first arginine (R5) in the twin arginine signal sequence of FdnG for a variety of other amino acids resulted in a dramatic (up to 60-fold) increase in the levels of protein synthesized. Bioinformatic analysis suggested that the mRNA specifying the first 17 codons of fdnG forms a stable stem-loop structure. A detailed mutational analysis has demonstrated the importance of this mRNA stem-loop in modulating FDH-N translation.

scholarly journals Identification of a cis-Acting Replication Element within the Poliovirus Coding Region

2000 ◽  
Vol 74 (10) ◽  
pp. 4590-4600 ◽  
Author(s):  
Ian Goodfellow ◽  
Yasmin Chaudhry ◽  
Andrew Richardson ◽  
Janet Meredith ◽  
Jeffrey W. Almond ◽  
...  
Keyword(s):  
Rna Virus ◽  
Site Directed Mutagenesis ◽  
Evolutionary Significance ◽  
Rna Structures ◽  
Coding Region ◽  
Rna Sequences ◽  
Stem Loop ◽  
Cis Acting ◽  
Function Recovery ◽  
Positive Sense

ABSTRACT The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel polioviruscis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

scholarly journals An intranasal ASO therapeutic targeting SARS-CoV-2

2021 ◽  
Author(s):  
Chi Zhu ◽  
Justin Y. Lee ◽  
Jia Z. Woo ◽  
Lei Xu ◽  
Xammy Nguyenla ◽  
...  
Keyword(s):  
Viral Replication ◽  
Locked Nucleic Acid ◽  
Rna Sequences ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Promising Therapeutic Approach ◽  
Vaccine Distribution ◽  
Rapid Deployment

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identified an LNA ASO binding to the 5′ leader sequence of SARS-CoV-2 ORF1a/b that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the K18-hACE2 humanized COVID-19 mouse model potently (98-99%) suppressed viral replication in the lungs of infected mice, revealing strong prophylactic and treatment effects. We found that the LNA ASO also represses viral infection in golden Syrian hamsters, and is highly efficacious in countering all SARS-CoV-2 "variants of concern" tested in vitro and in vivo, including B.1.427, B.1.1.7, and B.1.351 variants. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce transmission of variants partially resistant to vaccines and monoclonal antibodies, and could be deployed intranasally for prophylaxis or via lung delivery by nebulizer to decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences, and they may have particular impact in areas where vaccine distribution is a challenge, and could be stockpiled for future coronavirus pandemics.

scholarly journals The Red Clover Necrotic Mosaic Virus RNA2 trans-Activator Is Also a cis-Acting RNA2 Replication Element

2005 ◽  
Vol 79 (2) ◽  
pp. 978-986 ◽  
Author(s):  
Masahiro Tatsuta ◽  
Hiroyuki Mizumoto ◽  
Masanori Kaido ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Rna Synthesis ◽  
Red Clover ◽  
Loop Structure ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Protein Coding ◽  
Subgenomic Rna ◽  
Stem Loop Structure ◽  
Rna Interaction

ABSTRACT The expression of the coat protein gene requires RNA-mediated trans-activation of subgenomic RNA synthesis in Red clover necrotic mosaic virus (RCNMV), the genome of which consists of two positive-strand RNAs, RNA1 and RNA2. The trans-acting RNA element required for subgenomic RNA synthesis from RNA1 has been mapped previously to the protein-coding region of RNA2, whereas RNA2 is not required for the replication of RNA1. In this study, we investigated the roles of the protein-coding region in RNA2 replication by analyzing the replication competence of RNA2 mutants containing deletions or nucleotide substitutions. Our results indicate that the same stem-loop structure (SL2) that functions as a trans-activator for RNA-mediated coat protein expression is critically required for the replication of RNA2 itself. Interestingly, however, disruption of the RNA-RNA interaction by nucleotide substitutions in the region of RNA1 corresponding to the SL2 loop of RNA2 does not affect RNA2 replication, indicating that the RNA-RNA interaction is not required for RNA2 replication. Further mutational analysis showed that, in addition to the stem-loop structure itself, nucleotide sequences in the stem and in the loop of SL2 are important for the replication of RNA2. These findings suggest that the structure and nucleotide sequence of SL2 in RNA2 play multiple roles in the virus life cycle.

scholarly journals Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3′-Terminal Sequences of Viral Positive- and Negative-Strand RNA

2001 ◽  
Vol 75 (4) ◽  
pp. 1708-1721 ◽  
Author(s):  
Rajeev Banerjee ◽  
Asim Dasgupta
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Specific Interaction ◽  
Helicase Domain ◽  
Loop Structure ◽  
Stem Loop ◽  
Negative Strand ◽  
Stem Loop Structure ◽  
Rna Interaction ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and ΔpNS3 (helicase domain only) and examined their abilities to interact with the 3′-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3′ untranslated region (UTR). The results presented here demonstrate that NS3 (and ΔpNS3) interacts efficiently and specifically with the 3′-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5′-terminal RNA sequences. The interaction of NS3 with the 3′-terminal negative strand [called 3′(−) UTR127] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3′ terminus (nucleotides 5 to 20 from the 3′ end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and ΔpNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3′-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3′(−) UTR binding, specific interaction of NS3 (or ΔpNS3) with the 3′-terminal sequences of the positive-strand RNA [3′(+) UTR] appears to require the entire 3′(+) UTR of HCV. Deletion of either the 98-nucleotide 3′-terminal conserved region or the 5′ half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3′-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.

A predicted stem-loop in coat protein-coding sequencing of tobacco vein banding mosaic virus is required for efficient replication

2021 ◽  
Author(s):  
Zhi-Yong Yan ◽  
Le Fang ◽  
Xiao-Jie Xu ◽  
De-Jie Cheng ◽  
Cheng-Ming Yu ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Spontaneous Mutant ◽  
Loop Structure ◽  
Base Pairs ◽  
Stem Loop ◽  
Synonymous Substitutions ◽  
Coding Sequence ◽  
Stem Loop Structure ◽  
Viral Accumulation

Potyviral Coat protein (CP) is involved in the replication and movement of potyviruses. However, little information is available on the roles of CP-coding sequence in potyviral infection. Here, we introduced synonymous substitutions to the codon c574g575c576 coding conserved residue arginine at position 192 (R192) of tobacco vein banding mosaic virus (TVBMV) CP. Substitution of the codon c574g575c576 to a574g575a576 or a574g575g576, but not c574g575a576, c574g575t576, or c574g575g576, reduced the replication, cell-to-cell movement, and accumulation of TVBMV in Nicotiana benthamiana plants, suggesting that c574 was critical for replication of TVBMV. Nucleotides 531 to 576 of the TVBMV CP-coding sequence were predicted to form a stem-loop structure, in which four consecutive c-g base pairs (C576-G531, c532-g575, c574-g533, and C534-G573) were located at the stem. Synonymous substitutions of R178-codon c532g533c534 to A532G533A534 and A532G533G534, but not c532g533a534, c532g533t534, or c532g533g534, reduced the replication levels, cell-to-cell, and systemic movement of TVBMV, suggesting that c532 was critical for TVBMV replication. Synonymous substitutions disrupting base pairs C576-G531 and C534-G573 did not affect viral accumulation. After three serial passage inoculation, the accumulation of spontaneous mutant viruses was restored and codons A532G533A534, A532G533G534, a574g575a576, or a574g575g576 of mutants was separately changed to C532G533A534, C532G533G534, C574g575a576, or C574g575g576. Synonymous mutation of R178 and R192 also reduced viral accumulation in N. tabacum plants. Therefore, we concluded that the two consecutive c532-g575 and c574-g533 base pairs played critical roles in TVBMV replication via maintaining the stability of stem-loop structure formed by nucleotides 531 to 576 of CP-coding sequence.

scholarly journals trp RNA-Binding Attenuation Protein-5′ Stem-Loop RNA Interaction Is Required for Proper Transcription Attenuation Control of the Bacillus subtilis trpEDCFBAOperon

2000 ◽  
Vol 182 (7) ◽  
pp. 1819-1827 ◽  
Author(s):  
Hansen Du ◽  
Alexander V. Yakhnin ◽  
Subramanian Dharmaraj ◽  
Paul Babitzke
Keyword(s):  
Bacillus Subtilis ◽  
Rna Structure ◽  
Rna Binding ◽  
Structure Mapping ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Expression Studies ◽  
Attenuation Mechanism ◽  
Transcription Attenuation ◽  
Rna Interaction

ABSTRACT The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBAoperon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5′ end of the trp leader transcript also plays a role in the transcription attenuation mechanism. The 5′ stem-loop increases the affinity of TRAP fortrp leader RNA. Results from RNA structure mapping experiments demonstrate that the 5′ stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5′ stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with trpE′-′lacZtranslational fusions demonstrate that TRAP-5′ stem-loop interaction is required for proper regulation of the trp operon. 3′ RNA boundary experiments indicate that the 5′ structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus, TRAP-5′ stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.

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