Chemical Synthesis of Circular RNAs with Phosphoramidate Linkages for Rolling‐Circle Translation

Circular RNA without a stop codon enables rolling circle translation. we carried out one-pot chemical synthesis of circular RNA from RNA fragments. The synthesized circular RNAs acted as translation templates, despite the presence of unnatural phosphoramidate linkages.

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

10.1101/2021.03.23.433163 ◽

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.

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

Journal of General Virology ◽

10.1099/vir.0.013425-0 ◽

2009 ◽

Vol 90 (12) ◽

pp. 3057-3065 ◽

Cited By ~ 14

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.

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Pervasive translation of circular RNAs driven by short IRES-like elements

10.1101/473207 ◽

2018 ◽

Cited By ~ 7

Author(s):

Xiaojuan Fan ◽

Yun Yang ◽

Chuyun Chen ◽

Zefeng Wang

Keyword(s):

Mass Spectrometry ◽

Mass Spectrometry Data ◽

Circular Rnas ◽

Translation Control ◽

Rapid Degradation ◽

Random Sequences ◽

Rolling Circle ◽

A Cell

AbstractAlthough some circular RNAs (circRNAs) were found to be translated through IRES-driven mechanism, the scope and functions of circRNA translation are unclear because endogenous IRESs are rare. To determine the prevalence and mechanism of circRNA translation, we developed a cell-based system to screen random sequences and identified 97 overrepresented hexamers that drive cap-independent circRNA translation. These IRES-like short elements are significantly enriched in endogenous circRNAs and sufficient to drive circRNA translation. We further identified multiple trans-acting factors that bind these IRES-like elements to initiate translation. Using mass-spectrometry data, hundreds of circRNA-coded peptides were identified, most of which have low abundance due to rapid degradation. As judged by mass-spectrometry, 50% of translatable endogenous circRNAs undergo rolling circle translation, several of which were experimentally validated. Consistently, mutations of the IRES-like element in one circRNA reduced its translation. Collectively, our findings suggest a pervasive translation of circRNAs, providing profound implications in translation control.

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isoCirc catalogs full-length circular RNA isoforms in human transcriptomes

Nature Communications ◽

10.1038/s41467-020-20459-8 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Ruijiao Xin ◽

Yan Gao ◽

Yuan Gao ◽

Robert Wang ◽

Kathryn E. Kadash-Edmondson ◽

...

Keyword(s):

Rolling Circle Amplification ◽

Circular Rna ◽

Human Cell Line ◽

Full Length ◽

Circular Rnas ◽

Rna Seq ◽

Short Read ◽

Rolling Circle ◽

Rna Molecules ◽

Long Read

AbstractCircular RNAs (circRNAs) have emerged as an important class of functional RNA molecules. Short-read RNA sequencing (RNA-seq) is a widely used strategy to identify circRNAs. However, an inherent limitation of short-read RNA-seq is that it does not experimentally determine the full-length sequences and exact exonic compositions of circRNAs. Here, we report isoCirc, a strategy for sequencing full-length circRNA isoforms, using rolling circle amplification followed by nanopore long-read sequencing. We describe an integrated computational pipeline to reliably characterize full-length circRNA isoforms using isoCirc data. Using isoCirc, we generate a comprehensive catalog of 107,147 full-length circRNA isoforms across 12 human tissues and one human cell line (HEK293), including 40,628 isoforms ≥500 nt in length. We identify widespread alternative splicing events within the internal part of circRNAs, including 720 retained intron events corresponding to a class of exon-intron circRNAs (EIciRNAs). Collectively, isoCirc and the companion dataset provide a useful strategy and resource for studying circRNAs in human transcriptomes.

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Advances in Viroid-Host Interactions

Annual Review of Virology ◽

10.1146/annurev-virology-091919-092331 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Beatriz Navarro ◽

Ricardo Flores ◽

Francesco Di Serio

Keyword(s):

Infectious Agent ◽

Defense Responses ◽

Future Research ◽

Circular Rnas ◽

Annual Review ◽

Publication Date ◽

Structural Elements ◽

Rolling Circle ◽

Host Interactions ◽

Infectious Cycle

Viroids are small, single-stranded, circular RNAs infecting plants. Composed of only a few hundred nucleotides and being unable to code for proteins, viroids represent the lowest level of complexity for an infectious agent, even below that of the smallest known viruses. Despite the relatively small size, viroids contain RNA structural elements embracing all the information needed to interact with host factors involved in their infectious cycle, thus providing models for studying structure-function relationships of RNA. Viroids are specifically targeted to nuclei (family Pospiviroidae) or chloroplasts (family Avsunviroidae), where replication based on rolling-circle mechanisms takes place. They move locally and systemically through plasmodesmata and phloem, respectively, and may elicit symptoms in the infected host, with pathogenic pathways linked to RNA silencing and other plant defense responses. In this review, recent advances in the dissection of the complex interplay between viroids and plants are presented, highlighting knowledge gaps and perspectives for future research. Expected final online publication date for the Annual Review of Virology, Volume 8 is September 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Viroids and Viroid-like Circular RNAs: Do They Descend from Primordial Replicators?

Life ◽

10.3390/life12010103 ◽

2022 ◽

Vol 12 (1) ◽

pp. 103

Author(s):

Benjamin D. Lee ◽

Eugene V. Koonin

Keyword(s):

Plant Pathogens ◽

Rna World ◽

Unit Length ◽

Circular Rna ◽

Circular Rnas ◽

Rolling Circle ◽

Rna Molecules ◽

Evolution Of Life ◽

Rolling Circle Mechanism ◽

Viral Nucleocapsid

Viroids are a unique class of plant pathogens that consist of small circular RNA molecules, between 220 and 450 nucleotides in size. Viroids encode no proteins and are the smallest known infectious agents. Viroids replicate via the rolling circle mechanism, producing multimeric intermediates which are cleaved to unit length either by ribozymes formed from both polarities of the viroid genomic RNA or by coopted host RNAses. Many viroid-like small circular RNAs are satellites of plant RNA viruses. Ribozyviruses, represented by human hepatitis delta virus, are larger viroid-like circular RNAs that additionally encode the viral nucleocapsid protein. It has been proposed that viroids are direct descendants of primordial RNA replicons that were present in the hypothetical RNA world. We argue, however, that much later origin of viroids, possibly, from recently discovered mobile genetic elements known as retrozymes, is a far more parsimonious evolutionary scenario. Nevertheless, viroids and viroid-like circular RNAs are minimal replicators that are likely to be close to the theoretical lower limit of replicator size and arguably comprise the paradigm for replicator emergence. Thus, although viroid-like replicators are unlikely to be direct descendants of primordial RNA replicators, the study of the diversity and evolution of these ultimate genetic parasites can yield insights into the earliest stages of the evolution of life.

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Rolling Circle RNA Synthesis Catalysed by RNA

10.1101/2021.11.30.470609 ◽

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.

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