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PubMed1996

During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping.

Base Sequence↗

PubMed1988

Novel splicing mechanism for the ribosomal RNA intron in the archaebacterium Desulfurococcus mobilis.

The intron of the 23S rRNA gene of D. mobilis is excised from the pre-23S RNA at specific sites in vivo and subsequently ligated to form a stable circular RNA, with a normal 5'-3' phosphodiester bond, containing the entire intron sequence; 95% of this RNA codes for a protein of 194 amino acids that can be expressed in E. coli. Crude cell extracts from D. mobilis also induce a two-step slicing reaction in vitro, producing the same circular intron RNA but a low yield of ligated exons. Cleavage depends on the RNA structure adjacent to the cleavage site and yields a 3'-terminal phosphate. Splicing is enhanced by GTP, but does not require divalent metal ions. The cleavage and exon-splicing reactions resemble those found for tRNA introns in eukaryotes and a possible structural rationale for this similarity is considered together with its possible implications for the origin of eukaryotic rRNA and tRNA introns.

Amino Acid Sequence↗

PubMed2025

Comprehensive circRNA expression profile and hub genes screening during human liver development.

BACKGROUND: Understanding the expression of non-coding RNA in the liver during embryonic development provides important insights into liver diseases. Therefore, we investigated circular RNA (circRNA) roles in human liver development, an unexplored research domain. METHODS: Using high-throughput sequencing and bioinformatics, we analysed foetal liver samples across developmental stages (7-20 weeks post-conception). Differentially expressed (DE) genes were identified and subjected to enrichment analysis using Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), and Disease Ontology (DO). Modular analysis was performed using the Search Tool for Retrieval of Interacting Genes (STRING), followed by construction of a protein-protein interaction (PPI) network using Cytoscape software. The key genes were screened using Molecular Complex Detection (MCODE). The mRNA levels of hub genes were validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: There were 645 DE circRNAs and 5,145 DE mRNAs between human livers at the three growth stages (HB, EH, and LH). It was found that the activity of circRNAs was boosted remarkably in the hepatoblastic stage. Enrichment analysis found they mainly involved in nervous system regulation of liver function, embryonic organ development and digestive system development. In addition, DE circRNAs were primarily involved in the PI3K-AKT, MAPK and calcium pathways, potentially contributing to adult liver diseases. Notably, only hsa_circ_001471 and novel_circ_017382 were simultaneously identified at all stages and were persistently downregulated. A co-expression regulatory network involving these circRNAs was established. Three hub genes (LGR5, FOXL1 and RSPO3) were identified from the PPI network of 167 genes and may play key roles in human liver development. The RT-qPCR validation results were in agreement with the sequencing data. CONCLUSIONS: Our findings provide the first insights into the roles and regulatory networks of circRNAs in human liver development, laying the groundwork for further investigations of molecular and signalling networks.

Humans↗

PubMed1986

Control of Rous sarcoma virus RNA translation and packaging by the 5' and 3' untranslated sequences.

A cytopathic mutant of Rous sarcoma virus-PrB was isolated and shown to have two large deletions, which result in the junction of Gag sequences in P27 to the 5' end of Env, and in the loss of the Src gene. This replication-defective (rd) and transformation-defective (td) mutant can replicate in the presence of its helper, which is also td, but the viral particles produced are poorly infectious. Most of the virions do not contain viral RNA, and the mutant RNA accumulates in infected cells, where it is poorly translated and packaged. Molecular clones of the mutant, of its helper and of a PrBtd strain were obtained in lambda-EMBL3, characterized, shown to be biologically active by transfection assays and sequenced. Nucleotide sequence comparisons indicate that the strong ribosome-binding site of Rous sarcoma virus RNA, responsible for the efficient RNA translation in vivo and in vitro, is mutated in PrB-(HM) mutant RNA; this causes the inhibition of RNA translation, as demonstrated by translation competition experiments using virus RNA made in vitro that carries the original or the mutated ribosome-binding site. In addition, an insertion present at the 3' end of both the mutant and the helper RNA, but absent in PrBtd RNA, is probably responsible for the inhibition of RNA packaging. Finally, these data are discussed in the light of a model of a 5'----3' Rous sarcoma virus RNA structure leading to a circular RNA molecule, which has implications in RNA translation, packaging and reverse transcription.

Autoradiography↗

PubMed1992

Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae.

Yeast RNA viruses include L-A (and its toxin-encoding satellites M1, M2, ...) and L-BC dsRNA viruses and the single-stranded replicons 20S RNA and 23S RNA. L-A has a single-segment 4.6-kb linear genome encoding a major coat protein (gag) and its RNA-dependent RNA polymerase (pol), the latter expressed as a gag-pol fusion protein formed by a -1 ribosomal frameshift. In vitro replication, transcription, and binding systems for L-A have been used to define cis sites necessary for packaging and replication of viral RNA. Cellular functions that promote viral replication include the MAK3-encoded N-acetyltransferase whose modification of the gag N terminus is necessary for L-A virus assembly. The toxins encoded by the M satellite RNAs are processed by enzymes (KEX1 and KEX2, for killer expression) whose study led to discovery of mammalian hormone-processing enzymes. 20S RNA is an apparently naked circular RNA replicon (with a dsRNA form called W) encoding a RNA polymerase-like molecule. Its copy number is induced 10,000-fold in 1% potassium acetate, and it is subject to the same SKI antiviral system that represses L-A, L-BC, and M dsRNA copy number.

Base Sequence↗

PubMed2026

CircRNA-based CD19-targeted CAR-NK therapy for B-cell acute lymphoblastic Leukemia using a Coccidioides immitis-derived group II intron platform.

Chimeric antigen receptor (CAR)-T cell therapy targeting CD19 has demonstrated notable clinical efficacy in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), but its wider clinical applicability is constrained by long manufacturing processes, substantial costs, and severe adverse events. A potentially safer and more accessible alternative is provided by CAR-Natural killer (CAR-NK) cell therapy. Currently, most CAR-NK cells are generated using viral transduction, which is labor-intensive and associated with risks of genomic integration. Electroporation of CAR-encoding mRNA provides a non-integrating alternative but results in only transient CAR expression. Circular RNA (circRNA), owing to its enhanced stability and prolonged protein expression capacity, has recently emerged as a promising alternative to linear mRNA. To overcome the limitations of transient mRNA expression, we generated circRNA using a Group II intron-mediated cyclization system incorporating a newly selected Coccidioides immitis-derived Group II intron. The newly established Coccidioides immitis-derived Group II intron circularization system efficiently generated circRNA and supported more durable EGFP expression than linear mRNA in both HEK293T and NK92 cells. Using this system, we successfully developed a circRNA-based CD19-targeted CAR-NK platform. CircRNA-engineered CD19-targeted CAR-NK92 cells maintained more durable CAR expression and showed stronger antitumor activity at later time points. In mouse models of B-ALL, circRNA-engineered CAR-NK92 cells demonstrated better tumor control and extended survival compared with their linear mRNA-engineered counterparts. These results support the potential of circRNA-based CAR-NK therapy as an effective approach for enhancing the safety and efficacy of cancer immunotherapy.

Humans↗

PubMed1993

Circularly permuted DNA, RNA and proteins--a review.

Circular permutation represents a form of macromolecular isomerization when the normal termini are covalently linked and new termini introduced by breaking the backbone elsewhere. Here, we describe implications of circular permutation on the folding and function of biologically relevant macromolecules. A method permitting the analysis of the folding of all circularly permuted isomers of RNA is presented that has been successfully applied for a tRNA and the binding site of the coliphage R17 coat protein.

Animals↗

PubMed1994

Requirements for editing in the genomic RNA of hepatitis delta virus.

Hepatitis delta virus is a satellite of the hepatitis B virus which provides the surface antigen for the viral coat. The genome of the hepatitis delta virus consists of a single-stranded, circular RNA of 1679 nucleotides which forms a rod structure due to a high extent of self homology and which replicates via synthesis of an antigenomic RNA in a rolling circle mechanism similar to plant viroids. The antigenomic RNA contains the open reading frame for the delta-antigen which exists in two isoforms, p24 and p27. The formation of these two isoforms is explained by RNA editing at nucleotide 1012 which changes the stop translation codon UAG at amino acid residue 196 into the codon UGG for tryptophan and extends the open reading frame for the synthesis of p27. In order to investigate whether the editing occurs cotranscriptionally during RNA replication or is a posttranscriptional base modification in the genomic or antigenomic RNA, replication defective deletion mutants of the HDV genome were constructed and expressed in COS-7 cells. Editing was demonstrated in non-replicating fragments of genomic HDV RNA but not in antigenomic HDV RNA fragments. The sequences from nucleotide position 337-1200 of the genomic RNA were sufficient to enable low levels of editing. Editing at position 1012 required the opposite strand of the RNA rod from nucleotide position 337-783. Replicating circular HDV RNA was much more efficiently edited than non-replicating full length genomic HDV RNA. Expression of delta-antigen in trans did not complement the low editing efficiency of replication defective genomic HDV RNA. These results demonstrate posttranscriptional U to C editing in the genomic HDV RNA and exclude misincorporation during HDV RNA replication as the editing mechanism. The minimal structural requirements for HDV RNA editing reside between nucleotide position 337-1200.

Animals↗

PubMed1987

Self-cleavage of RNA in the replication of viroids and virusoids.

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.

Base Sequence↗

PubMed2004

Hepatitis delta virus antigen is methylated at arginine residues, and methylation regulates subcellular localization and RNA replication.

Hepatitis delta virus (HDV) contains a circular RNA which encodes a single protein, hepatitis delta antigen (HDAg). HDAg exists in two forms, a small form (S-HDAg) and a large form (L-HDAg). S-HDAg can transactivate HDV RNA replication. Recent studies have shown that posttranslational modifications, such as phosphorylation and acetylation, of S-HDAg can modulate HDV RNA replication. Here we show that S-HDAg can be methylated by protein arginine methyltransferase (PRMT1) in vitro and in vivo. The major methylation site is at arginine-13 (R13), which is in the RGGR motif of an RNA-binding domain. The methylation of S-HDAg is essential for HDV RNA replication, especially for replication of the antigenomic RNA strand to form the genomic RNA strand. An R13A mutation in S-HDAg inhibited HDV RNA replication. The presence of a methylation inhibitor, S-adenosyl-homocysteine, also inhibited HDV RNA replication. We further found that the methylation of S-HDAg affected its subcellular localization. Methylation-defective HDAg lost the ability to form a speckled structure in the nucleus and also permeated into the cytoplasm. These results thus revealed a novel posttranslational modification of HDAg and indicated its importance for HDV RNA replication. This and other results further showed that, unlike replication of the HDV genomic RNA strand, replication of the antigenomic RNA strand requires multiple types of posttranslational modification, including the phosphorylation and methylation of HDAg.

Amino Acid Sequence↗

PubMed1996

Inverse splicing of a discontinuous pre-mRNA intron generates a circular exon in a HeLa cell nuclear extract.

We have recently reported the first example of inverse splicing of a eukaryotic pre-mRNA intron using a whole cell extract from the yeast Saccharomyces cerevisiae. The concomitant circularization of the exon in the course of this splicing reaction gave rise to the hypothesis that the circular RNA species, which had been recently discovered in vivo in mammalian cells, were generated by inverse splicing. Here we report the formation of a circular exon in HeLa cell nuclear extracts by an inverse splicing reaction of the second intron of the human beta-globin gene from a pre-mRNA transcript in which the two intron halves flanked an artificially fused, single exon. Our data demonstrate that the mammalian pre-mRNA splicing system has indeed an intrinsic capability of aligning splice sites in reverse order and that this alignment can be followed by a complete splicing reaction, whereby the discontinuous intron sequences are removed. Thus we propose that circular exons in vivo arise as a result of an inverse splicing reaction following the pairing of a 5' splice site with an upstream 3' splice site and that the frequency of this event is influenced by the presence and strength of other, competing splice sites.

Cell Extracts↗

PubMed1985

Unusual properties of two branched RNA's with circular and linear components.

Irradiation with ultraviolet light was used to create two nonlinear RNA molecules. Circular potato spindle tuber viroid (PSTV) RNA was crosslinked at a single site to generate a figure eight-shaped molecule; 5S rRNA from HeLa cells was transformed into an alpha-shaped molecule with a small circular element and two arms (1). Crosslinked RNA's could be separated from their untreated counterparts by electrophoresis in polyacrylamide gels containing urea. The gel mobility of crosslinked PSTV was not altered by boiling, treatment with E. coli RNase III or glyoxalation. However, mild nuclease digestion ("nicking") produced derivatives which migrated more slowly than the starting material in gels of certain polyacrylamide concentrations, but not in others. Limited nuclease digestion of crosslinked 5S rRNA did not generate any detectable products with reduced mobility in the gels tested. Thus, the ability of the "nicking assay" to reveal circular elements within nonlinear RNA's can vary depending upon the composition of the gel chosen for analysis and on the size of the circular element relative to the rest of the molecule.

Electrophoresis, Polyacrylamide Gel↗

PubMed2006

HDV RNA replication: ancient relic or primer?

HDV replicates its circular RNA genome using a double rolling-circle mechanism and transcribes a hepatitis delta antigen-encodeing mRNA from the same RNA template during its life cycle. Both processes are carried out by RNA-dependent RNA synthesis despite the fact that HDV does not encode an RNA-dependent RNA polymerase (RdRP). Cellular RNA polymerase II has long been implicated in these processes. Recent findings, however, have shown that the syntheses of genomic and antigenomic RNA strands have different metabolic requirements, including sensitives to alpha-amanitin and the site of synthesis. Evidence is summarized here for the involvement of other cellular polymerases, probably pol I, in the synthesis of antigenomic RNA strand. The ability of mammalian cells to replicate HDV RNA implies that RNA-dependent RNA synthesis was preserved throughout evolution.

Hepatitis Delta Virus↗

PubMed2025

Expression regulation network in papillae of sea cucumbers: Whole-transcriptome and DNA methylation datasets.

To elucidate the expression regulation network of papilla size of sea cucumbers (Apostichopus japonicus), the whole-transcriptome and DNA methylome datasets of different sizes of papillae in sea cucumbers were generated. Average clean bases of whole-transcriptome (16.35 G) and DNA methylome (28.92 G) were obtained using RNA sequencing and whole-genome bisulfite sequencing techniques. A total of 3,188 ceRNA networks were also identified including 3,081 long non-coding RNAs (lncRNA)/microRNAs (miRNA)/mRNA networks and 107 circular RNA (circRNA)/miRNA/mRNA networks. Methylome data indicate that there were 3,307 and 3,776 differentially methylated regions (DMRs) with high-level methylation as well as 3,125 and 3,016 DMRs with low-level methylation in big papillae compared to small papillae. The identified DMRs were mainly distributed in introns, promotors, or exons. The whole-transcriptome and DNA methylome datasets generated from this study not only established a robust theoretical foundation (especially from the epigenetic aspect) for elucidating expression regulation network determining papilla size in sea cucumbers but also can be a valuable resource of biomarker mining for papilla appearance-based selective breeding in sea cucumbers.

DNA Methylation↗

PubMed2026

Beyond Canonical Neoantigens: Emerging Technologies for Identification of Noncanonical Antigens and Implications for Personalized Cancer Vaccines.

Over the past decade, advances in sequencing technologies and computational pipelines enabled the development of personalized cancer vaccines (PCVs). Current PCV strategies primarily target cancer neoantigens generated by non-synonymous DNA mutations, which can result in altered amino acid sequences capable of eliciting tumor-specific immune responses. More recently, a distinct class of tumor-specific antigens (TSA), termed noncanonical or cryptic antigens, has emerged as an additional source of immunogenic targets. Unlike canonical neoantigens, noncanonical antigens typically cannot be identified by tumor/normal whole-exome sequencing, as they do not arise from classical DNA mutations. Instead, they are often associated with less well recognized and/or aberrant processes in the pathways from DNA to human leukocyte antigen (HLA)-presented peptides. Examples include transposable elements, circular RNA, translation of alternative open reading frames and/or long non-coding RNA, among others. Emerging evidence suggests that noncanonical antigens represent a substantial portion of the tumor-specific immunopeptidome and, similar to canonical neoantigens, are absent during thymic selection and can evade central tolerance and elicit T cell responses. Technological advances have increasingly facilitated the identification of noncanonical antigens. Long-read RNA sequencing reveals noncanonical transcripts by improving transcriptome assembly, while ribosome profiling provides genome-wide maps of actively translated regions, facilitating the discovery of peptides from aberrant translation events. Specialized molecular approaches enable enrichment and sequencing of circular RNAs, and immunopeptidomics using mass spectrometry allows for direct characterization of HLA-presented peptides. Together, these technological advances have led to an increasing interest in prioritizing and targeting noncanonical antigens in the next generation of PCVs. This review provides an overview of the diverse origins of TSAs beyond classical neoantigens and discusses emerging approaches that may enable the integration of these antigens in future clinical trials.

circular RNA↗

PubMed2002

Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo.

Protein-RNA interactions play indispensable structural, catalytic, and regulatory roles within the cell. Understanding their physical association in vivo provides valuable insight into their assembly, function, and regulation in the cellular milieu. Inspired by the chromatin immunoprecipitation assay, we have developed a ribonucleoprotein (RNP) immunoprecipitation assay to study RNA-protein interactions in vivo. This method takes advantage of the highly reactive, reversible crosslinker formaldehyde, combined with high-stringency immunoprecipitation to identify specific RNAs associated with a given protein. The RNP immunoprecipitation (RIP) assay was developed using RNA-protein interactions of hepatitis delta virus (HDV) as a model system. HDV is an RNA virus with a single-stranded circular RNA genome that encodes one viral protein, hepatitis delta antigen (HDAg). The high affinity of HDAg for the HDV RNA genome, combined with the well-characterized anti-HDAg antibodies, made this system a logical starting point for the development of the RIP assay. Cells with replicating HDV were crosslinked with formaldehyde and the HDV RNPs were immunoprecipitated using anti-HDAg antibodies. The crosslinks were then reversed by heat treatment, and the immunoprecipitated HDV RNAs were identified by reverse transcription polymerase chain reaction (RT-PCR). The specificity of this assay was tested using HDV mutants and heterologous antibodies for immunoprecipiation followed by RT-PCR with HDV-specific primers. This experiment showed no nonspecific immunoprecipitation of the HDV RNPs. The method was tested further using protein-RNA interactions known to exist in the U1 snRNP. The results indicate that the RIP assay is a powerful tool to identify RNA-protein interactions in vivo and has the potential to unravel the cellular network of RNP complexes in their native setting.

Cross-Linking Reagents↗

PubMed2001

Animal models of hepatitis delta virus infection and disease.

Hepatitis delta virus (HDV) is a defective RNA virus with similarities to unusual subviral pathogens of higher plants. It requires hepatitis B virus (HBV) for its replication/transmission, and HBV-infected humans are the only established host. HDV causes both severe acute hepatitis and rapidly progressive chronic disease in some individuals. The HDV life cycle involves remarkable features, such as ribozyme- mediated autocatalytic processes, Pol II-directed RNA synthesis from a single-stranded circular RNA template, and RNA editing. Much of our understanding of the nature of this pathogen derives from experimental studies in the chimpanzee model of HBV infection. The hepadnavirus-infected eastern woodchuck also is capable of supporting HDV replication and offers opportunities for the development of control strategies that might be applicable to human type D hepatitis.

Acute Disease↗

PubMed1996

Exon circularization in mammalian nuclear extracts.

Correct ligation of exons in pre-mRNA splicing requires splice site juxtaposition (splice site pairing), usually involving a 5' splice site and a downstream 3' splice site. Splicing of a 5' splice site to an upstream 3' splice site, however, is predicted to result in a circular RNA. This mode of splice site pairing across the axon has been hypothesized to account for rare RNAs containing scrambled exons (Nigro JM et al., 1991, Celt 64:607-613; Cocquerelle C et al., 1992, EMBO J 11:1 095-1098). Additionally, this mode of splice site pairing has been postulated to explain the formation of SRY circular transcripts in mouse testis (Capel B et al., 1993, Celt 73:1019- 1030). Here we show that splice site pairing across the exon can result in exon circularization in vitro. These results indicate that spliceosome-mediated axon circularization indeed can account for the formation of scrambled exons and circular RNAs. Exon circularization efficiency decreased dramatically as the length of the exon was increased from 95 nt to 274 nt. Circularization of this longer exon was restored, however, when intronic complementary sequences were included in the RNA substrate. These complementary sequences could form a stem that served to bring the splice sites into proximity and thereby promote splice site pairing. Therefore, the splicing of this structured RNA recapitulated SRY-like exon circularization in vitro.

Base Sequence↗