Knockout of circRNAs by base editing back-splice sites of circularized exons
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA722401 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14252140.fastq.gz | Fastqsanger.gz | |||
| SRR14252141.fastq.gz | Fastqsanger.gz | |||
| SRR14252142.fastq.gz | Fastqsanger.gz | |||
| SRR14252143.fastq.gz | Fastqsanger.gz |
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Project description:Subcellular RNA-seq datasets are used for genome-wide analysis of circRNAs in 293FT cells. Here we knocked out circRNAs by base editor (BE)-mediated nucleotide changes.
2021-12-01 | GSE172193 | GEO
Project description:Many circular RNAs (circRNAs) are produced from back-splicing of exons of precursor mRNAs and are generally co-expressed with cognate linear RNAs. Methods for circRNA-specific knockout are lacking, largely due to sequence overlaps between forms. Here, we use base editors (BEs) for circRNA depletion. By targeting splice sites involved in both back-splicing and canonical splicing, BEs can repress circular and linear RNAs. Targeting sites predominantly for circRNA biogenesis, BEs could efficiently repress the production of circular but not linear RNAs. As hundreds of exons are predominantly back-spliced to produce circRNAs, this provides an efficient method to deplete circRNAs for functional study.
| S-EPMC8744252 | biostudies-literature
Project description:Conversely to canonical splicing, back-splicing covalently ligates the upstream 3' splice site (SS) with downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely-identified in eukaryotes and have regulatory functions in gene expression. However, sex-specific back-splicing in Drosophila has not been investigated and its regulation remains unclear. Here, we performed multiple RNA-seq of various sex-specific Drosophila samples including head, body and gonads from both genders, and identified more than ten thousand of circular RNAs, in which hundreds are sex-differentially expressed and back-spliced. Intriguingly, we found that expression of SXL, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene which only functionally spliced in females, promotes back-splicing of many female-differentially expressed circRNAs in the male S2 cells, while expression of a SXL mutant did not. Using a monoclonal antibody, we further obtained the transcriptome-wide RNA-binding sites of SXL through a PAR-CLIP approach and revealed that SXL-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing of those circRNAs, whereas SXL-binding on the circRNA exons inhibits the back-splicing. This study provides strong evidence that SXL has a regulatory role in back-splicing to generate sex-specifc circRNAs, as well as in the initiation of Drosophila sex-determination cascade through canoncial forward-splicing.
2023-04-23 | GSE221102 | GEO
Project description:Back-splice junctions of KSHV circular RNAs
| PRJNA694907 | ENA
Project description:Aberrant splice variants are involved in the initiation and/or progression of glial brain tumors. We therefore set out to identify splice variants that are differentially expressed between histological subgroups of gliomas. Splice variants were identified using a novel platform that profiles the expression of virtually all known and predicted exons present in the human genome. Exon-level expression profiling was performed on 26 glioblastomas, 22 oligodendrogliomas and 6 control brain samples. Our results demonstrate that Human Exon arrays can identify subgroups of gliomas based on their histological appearance and genetic aberrations. We next used our expression data to identify differentially expressed splice variants. In two independent approaches, we identified 49 and up to 459 exons that are differentially spliced between glioblastomas and oligodendrogliomas a subset of which (47% and 33%) were confirmed by RT-PCR. In addition, exon-level expression profiling also identified >700 novel exons. Expression of ~67% of these candidate novel exons was confirmed by RT-PCR. Our results indicate that exon-level expression profiling can be used to molecularly classify brain tumor subgroups, can identify differentially regulated splice variants and can identify novel exons. The splice variants identified by exon-level expression profiling may help to detect the genetic changes that cause or maintain gliomas and may serve as novel treatment targets. Keywords: cell type comparison 6 adult non diseased brain, 26 glioblastomas, 21 oligodendrogliomas
2008-01-28 | E-GEOD-9385 | biostudies-arrayexpress
Project description:Aberrant splice variants are involved in the initiation and/or progression of glial brain tumors. We therefore set out to identify splice variants that are differentially expressed between histological subgroups of gliomas. Splice variants were identified using a novel platform that profiles the expression of virtually all known and predicted exons present in the human genome. Exon-level expression profiling was performed on 26 glioblastomas, 22 oligodendrogliomas and 6 control brain samples. Our results demonstrate that Human Exon arrays can identify subgroups of gliomas based on their histological appearance and genetic aberrations. We next used our expression data to identify differentially expressed splice variants. In two independent approaches, we identified 49 and up to 459 exons that are differentially spliced between glioblastomas and oligodendrogliomas a subset of which (47% and 33%) were confirmed by RT-PCR. In addition, exon-level expression profiling also identified >700 novel exons. Expression of ~67% of these candidate novel exons was confirmed by RT-PCR. Our results indicate that exon-level expression profiling can be used to molecularly classify brain tumor subgroups, can identify differentially regulated splice variants and can identify novel exons. The splice variants identified by exon-level expression profiling may help to detect the genetic changes that cause or maintain gliomas and may serve as novel treatment targets. Keywords: cell type comparison
2008-01-29 | GSE9385 | GEO
Project description:Circular RNAs (circRNAs) are a class of ubiquitously expressed, single-stranded, covalently covalently-closed (i.e. circularised) RNA that contain a unique nucleotide sequence created by the ligation of their 5' and 3' ends, called the back-splice junction. Understanding the cellular roles of circRNAs involves, in part, investigating the effects on cell phenotype of increased expression of individual circRNAs. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote circularisation. We observed that all commonly used plasmids we tested unexpectedly incorporated molecular scars comprising vector sequence vector sequence as a molecular scar into the circRNA back-splice junction upon circularisation. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural back-splice junction at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that enable selection with a variety of antibiotics and fluorescent proteins, a range of promoters varying in promoter strength and plus a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfil a critical need for the investigation of circRNA function, including the role of the unique back-splice junction.
2023-12-14 | GSE246020 | GEO
Project description:Epigenetically-controlled tumor antigens derived from splice junctions between exons and transposable elements
| PRJNA902299 | ENA
Project description:In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17-188 fold relative to nucleated tissues, and 14-26 fold relative to samples digested with RNAseR to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched ~13X in platelets relative to nucleated tissues, and identify 3162 genes significantly enriched for circRNAs including some where all RNAs appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in megakaryocytes, and that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost on aveage over 90% of their progenitor mRNAs, and that translation in platelets occurrs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artefacts. A single rRNA depleted total RNA sample was sequenced. This together with 25 publicly available rRNA depleted total RNA samples (including 3 from platelets) were analysed using PTESFinder v 1 (http://sourceforge.net/projects/ptesfinder-v1/) to identify back-splice junctions, characteristic of circRNA transcripts. The contribution of circRNA producing exons was analysed on a gene by gene basis as follows: All circRNA transcripts identified in any sample were first pooled to define exons which can contribute to circRNA generation using custom scripts (available on request). For each sample, expression estimates (RPKMI) across all circRNA producing exons were computed for each locus using the total size of exons (in bp) and the read counts mapping to them. Similarly, total size and exonic read counts for exons for which no circRNA were detected in any sample were used to compute expression estimates (RPKME) for non-circRNA producing exons for each locus. Abundance ratios (RPKMI/RPKME and RPKMI/RPKMI+RPKME) were calculated and compared between Platelets and human tissues using Wilcoxon signed-rank test. Please note that the '25sample_info_accn_no.txt' contains the accession numbers and tissue/cell type information for 25 samples analyzed together.
2015-12-12 | E-GEOD-69192 | biostudies-arrayexpress
Project description:To explore the overall circRNAs involved in growth and development of Arabidopsis thaliana across the lifespan, we deeply sequenced samples of whole plants from different developmental stages (cotyledons emergence, rosette leavesï¹¥1 mm, rosette growth complete, first flower open, flourishing florescence, first silique shattered, senescence). The total RNA was purified by rRNA-depletion and linear RNA removal with RNAseR, and sequenced by the Illumina HiSeq2500 platform. We obtained 31 Gb raw data and identified 1217 circRNAs with expression quantity. We annotated these circRNAs and predicted their targeted microRNA. The circRNAs involved in growth and development of Arabidopsis thaliana across lifespan were identified and analyzed using the Illumina HiSeq2500 platform.
2016-09-01 | E-GEOD-77672 | biostudies-arrayexpress