scholarly journals Rolling Circle RNA Synthesis Catalysed by RNA

2021 ◽  
Author(s):  
Emil Laust Kristoffersen ◽  
Matthew Burman ◽  
Agnes Noy ◽  
Philipp Holliger
Keyword(s):  
Rna Synthesis ◽  
Hammerhead Ribozyme ◽  
Rna Replication ◽  
Genetic System ◽  
Circular Rnas ◽  
Potential Solution ◽  
Strand Displacement ◽  
Separation Problem ◽  
Rolling Circle ◽  
Dynamics Simulations

RNA-catalysed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5′ and 3′ ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

scholarly journals Mutational analysis of eggplant latent viroid RNA processing in Chlamydomonas reinhardtii chloroplast

2009 ◽  
Vol 90 (12) ◽  
pp. 3057-3065 ◽  
Author(s):  
Fernando Martínez ◽  
Jorge Marqués ◽  
María L. Salvador ◽  
José-Antonio Daròs
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Mutational Analysis ◽  
Hammerhead Ribozyme ◽  
Experimental System ◽  
Circular Rnas ◽  
Rolling Circle ◽  
Transcript Processing ◽  
Transcript Cleavage ◽  
The Right

Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational analysis of sequence and structural elements in the ELVd molecule that are involved in transcript processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three deletion ELVd mutants was created and expressed in C. reinhardtii chloroplast. All mutants cleaved efficiently except for the control with an insertion inside the hammerhead ribozyme domain, supporting the prediction that this domain is necessary and sufficient to mediate transcript cleavage in vivo. However, two deletion mutants that cleaved efficiently showed ligation defects, indicating that during RNA circularization, other parts of the molecule are involved in addition to the hammerhead ribozyme domain. This is probably a quasi double-stranded structure present in the central part of the molecule which contains the ligation site in an internal loop. However, the mutations prevented the viroid from infecting its natural host, eggplant, indicating that they affected other essential functions in ELVd infectious cycle. The insertion in the terminal loop of the right upper hairpin of ELVd did not have this effect; it was tolerated and partially maintained in the progeny.

scholarly journals Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases

2002 ◽  
Vol 76 (8) ◽  
pp. 3920-3927 ◽  
Author(s):  
Thomas B. Macnaughton ◽  
Stephanie T. Shi ◽  
Lucy E. Modahl ◽  
Michael M. C. Lai
Keyword(s):  
Rna Polymerase Ii ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Rna Polymerases ◽  
Rolling Circle Replication ◽  
Hepatitis Delta ◽  
Rolling Circle ◽  
System L ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [32P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40°C and becoming virtually undetectable at temperatures below 30°C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 μg/ml) of α-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by α-amanitin at concentrations as low as 2.5 μg/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.

scholarly journals Multiple genomic sequences of hepatitis delta virus are associated with cDNA promoter activity and RNA double rolling-circle replication

2012 ◽  
Vol 93 (3) ◽  
pp. 577-587 ◽  
Author(s):  
Fu-Tien Liao ◽  
Li-Sung Hsu ◽  
Jiunn-Liang Ko ◽  
Chun-Che Lin ◽  
Gwo-Tarng Sheu
Keyword(s):  
Hepatitis Delta Virus ◽  
Promoter Activity ◽  
Rna Synthesis ◽  
Stop Codon ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Rolling Circle ◽  
Delta Virus

To understand how DNA-dependent RNA polymerase II (pol II) recognizes hepatitis delta virus (HDV) RNA as a template, it is first necessary to identify the HDV sequence that acts as a promoter of pol II-initiated RNA synthesis. Therefore, we isolated the pol II-response element from HDV cDNA and examined the regulation by hepatitis delta antigens (HDAgs). Two HDV cDNA fragments containing bidirectional promoter activity were identified. One was located at nt 1582–1683 (transcription-promoter region 1, TR-P1) and the other at nt 1223–1363 (transcription-internal region 5, TR-I5). The promoter activities of these two regions were enhanced by HDAgs to differing degrees. Next, the role of these sequences in an HDV cDNA-free RNA replication system was characterized by site-directed mutagenesis. Our data showed that: (i) the AUG codon at the HDAg ORF of HDV RNA (nt 1599–1601) that mutates to UAG (amber stop codon) results in loss of dimeric but not monomeric HDV RNA synthesis. (ii) A 5 nt mutation of TR-P1 (P1-m5, nt 1670–1674) abolishes RNA replication completely. Two-nucleotide-mutated RNA (P1-m2, nt 1662–1663) is able to synthesize short RNAs but not monomeric HDV RNA. (iii) A mutation in 5 nt at the TR-I5 region (I5-m5, nt 1351–1355) also abolishes HDV replication. Mutants with 2 nt mutations (I5-m2, nt 1351–1352) or 3 nt mutations (I5-m3, nt 1353–1355) inhibit HDV dimeric but not monomeric RNA synthesis. Furthermore, large HDAg is expressed in cells transfected with I5-m3 and I5-m2 RNAs and that demonstrate the RNA-editing event in the monomeric HDV RNA. These results provide further understanding of the double rolling-circle mechanism in HDV RNA replication.

Fluorometric determination of microRNA based on strand displacement amplification and rolling circle amplification

2017 ◽  
Vol 184 (11) ◽  
pp. 4359-4365 ◽  
Author(s):  
Yunlei Zhou ◽  
Bingchen Li ◽  
Minghui Wang ◽  
Jun Wang ◽  
Huanshun Yin ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Strand Displacement ◽  
Fluorometric Determination ◽  
Strand Displacement Amplification ◽  
Rolling Circle

scholarly journals Enzymatic RNA Production from NTPs Synthesized from Nucleosides and Trimetaphosphate

2021 ◽  
Author(s):  
Fabio Chizzolini ◽  
Alexandra Kent ◽  
Luiz F. M. Passalacqua ◽  
Andrej Lupták
Keyword(s):  
Rna Synthesis ◽  
Rna World ◽  
Rna Replication ◽  
High Energy ◽  
Rna Degradation ◽  
T7 Rna Polymerase ◽  
Nucleotide Synthesis ◽  
Enthalpy Of Activation ◽  
Rna Polymerization ◽  
Synthetic Approaches

<p>A mechanism of nucleoside triphosphorylation would have been critical in an evolving “RNA world” to provide high-energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphoates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. We demonstrate that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under mild, prebiotically-relevant conditions, with second-order rate constants ranging from 1.7 x 10<sup>–6</sup> to 6.5 x 10<sup>–6</sup> M<sup>–1</sup> s<sup>–1</sup>. The ATP reaction shows a linear dependence on pH and Mg<sup>2+</sup>, and an enthalpy of activation of 88 ± 4 kJ/mol. At millimolar nucleoside and cTmp concentrations, the rNTP production rate is sufficient to facilitate RNA synthesis by both T7 RNA polymerase and a polymerase ribozyme. We suggest that the optimized reaction of cTmp with nucleosides may provide a viable connection between prebiotic nucleotide synthesis and RNA replication.</p>

scholarly journals Overexpression-based detection of translatable circular RNAs is vulnerable to coexistent linear RNA byproducts

2021 ◽  
Author(s):  
Yanyi Jiang ◽  
Xiaofan Chen ◽  
Wei Zhang
Keyword(s):  
Open Reading Frames ◽  
Systematic Evaluation ◽  
Circular Rnas ◽  
Protein Coding ◽  
Rolling Circle ◽  
Functional Investigation ◽  
Overexpression System ◽  
Translation Signals ◽  
Coding Potential ◽  
Reading Frames

AbstractIn RNA field, the demarcation between coding and non-coding has been negotiated by the recent discovery of occasionally translated circular RNAs (circRNAs). Although absent of 5’ cap structure, circRNAs can be translated cap-independently. Complementary intron-mediated overexpression is one of the most utilized methodologies for circRNA research but not without bearing echoing skepticism for its poorly defined mechanism and latent coexistent side products. In this study, leveraging such circRNA overexpression system, we have interrogated the protein-coding potential of 30 human circRNAs containing infinite open reading frames in HEK293T cells. Surprisingly, pervasive translation signals are detected by immunoblotting. However, intensive mutagenesis reveals that numerous translation signals are generated independently of circRNA synthesis. We have developed a dual tag strategy to isolate translation noise and directly demonstrate that the fallacious translation signals originate from cryptically spliced linear transcripts. The concomitant linear RNA byproducts, presumably concatemers, can be translated to allow pseudo rolling circle translation signals, and can involve backsplicing junction (BSJ) to disqualify the BSJ-based evidence for circRNA translation. We also find non-AUG start codons may engage in the translation initiation of circRNAs. Taken together, our systematic evaluation sheds light on heterogeneous translational outputs from circRNA overexpression vector and comes with a caveat that ectopic overexpression technique necessitates extremely rigorous control setup in circRNA translation and functional investigation.

scholarly journals Viroids: unusual small pathogenic RNAs.

2004 ◽  
Vol 51 (3) ◽  
pp. 587-607 ◽  
Author(s):  
Anna Góra-Sochacka
Keyword(s):  
Secondary Structure ◽  
Hammerhead Ribozyme ◽  
Direct Interaction ◽  
Plant Diseases ◽  
Plant Host ◽  
Rolling Circle ◽  
Rna Molecules ◽  
Viroid Replication ◽  
Plant Genes ◽  
Rolling Circle Mechanism

Viroids are small (about 300 nucleotides), single-stranded, circular, non-encapsidated pathogenic RNA molecules. They do not code for proteins and thus depend on plant host enzymes for their replication and other functions. They induce plant diseases by direct interaction with host factors but the mechanism of pathogenicity is still unknown. They can alter the expression of selected plant genes important for growth and development. Viroids belong to two families, the Avsunviroidae and the Pospiviroidae. Viroids of the Avsunviroidae family adopt a branched or quasi rod-like secondary structure in their native state. Members of the Pospiviroidae family adopt a rod-like secondary structure. In such native structures five structural/functional domains have been identified: central (C), pathogenicity, variable and two terminal domains. The central conserved region (CCR) within the C domain characterizes viroids of the Pospiviroidae. Specific secondary structures of this region play an important role in viroid replication and processing. Viroids of the Avsunviroidae family lack a CCR but possess self-cleaving properties by forming hammerhead ribozyme structures; they accumulate and replicate in chloroplasts, whereas members of the Pospiviroidae family have a nuclear localization. Viroid replication occurs via a rolling circle mechanism using either a symmetric or asymmetric pathway in three steps, RNA transcription, processing and ligation.

scholarly journals The Large Delta Antigen of Hepatitis Delta Virus Potently Inhibits Genomic but Not Antigenomic RNA Synthesis: a Mechanism Enabling Initiation of Viral Replication

2000 ◽  
Vol 74 (16) ◽  
pp. 7375-7380 ◽  
Author(s):  
Lucy E. Modahl ◽  
Michael M. C. Lai
Keyword(s):  
Life Cycle ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Dominant Negative ◽  
Genomic Rna ◽  
Inhibitory Effects ◽  
Hepatitis Delta ◽  
Artificial Dna ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.

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