Ontology highlight
ABSTRACT:
PROVIDER: PRJNA647596 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12284170.fastq.gz | Fastqsanger.gz | |||
| SRR12284171.fastq.gz | Fastqsanger.gz | |||
| SRR12284172.fastq.gz | Fastqsanger.gz | |||
| SRR12284173.fastq.gz | Fastqsanger.gz | |||
| SRR12284174.fastq.gz | Fastqsanger.gz |
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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:The MS/MS spectra were searched against a custom protein database from span back-splice junction circRNA open reading frame prediction.
2020-08-21 | PXD021030 |
Project description:Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5’ cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs. We increased the analytical stringency of our workflow by retaining peptides identified independently by two mass spectrometry search engines and/or by applying a group-specific FDR for canonical-derived and circRNA-derived peptides. A subset of circRNA-derived peptides specifically encoded by the region spanning the back-splice junction (BSJ) were validated with targeted MS, and with direct Sanger sequencing of the respective source transcripts. Our workflow identified 54 unique BSJ-spanning circRNA-derived peptides in the immunopeptidome of melanoma and lung cancer samples. Our novel approach enlarges the catalog of source proteins that can be explored for immunotherapy.
2024-03-20 | PXD043989 | Pride
Project description:Circular RNAs are a class of non-coding RNAs mostly generated by back-splicing RNA transcripts of protein-coding gene loci. In difference to canoncial splicing, back-splicing ligates downstream splice donor site with an upstream splice acceptor generating a covalently closed circular RNA. CircRNAs have been reported to influence cellular functions by acting as miRNA sponges, regulation of host gene transcription or by protein interactions. The circRNA cZNF292 has been previously reported to be regulated under hypoxia and to influence endothelial cell angiogenesis and functions. The present data set aims to identfy protein interactions partners of cZNF292 in endothelial cells to further characterize the mechanism of action of cZNF292. Dataset comprises 8 replicates of proteins identified after biotin-based 2'O-Me RNA antisense affinity purification of the circRNA cZNF292 from HUVEC (human umbilical vein endothelial cell) lysates compared to a control probe dataset. Purification was performed without cross-linking under low salt and mild detergent conditions.
2022-11-29 | PXD029794 | Pride
Project description:To determine the circRNA expression profile in C2C12 myoblasts and myotubes, we used mouse circRNA microarray from Arraystar to examine the expression of circRNAs in C2C12 myoblasts and myotubes.
2021-03-19 | GSE112014 | GEO
Project description:Circular RNAs (circRNAs) constitute an abundant class of covalently closed non-coding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. Here, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We identified 5807 circRNAs in total RNA sequencing data from 4 liver cancer cell lines and used the back splice junction (BSJ) sequences of 7 validated circRNAs as target sites for designing different LNA-modified ASOs for circRNA knockdown. We found that while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript and may also cause degradation of unintended off-targets. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs or LNA/DNA mixmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes or predicted off-targets. In summary, our results demonstrate that LNA-modified ASOs complementary to BSJ sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.
2022-11-01 | GSE145592 | GEO
Project description:Many new alternative splice forms have been detected at the transcript level using next generation sequencing (NGS) methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of NGS, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splice-junction peptides. Results: Eighty million paired-end Illumina reads and ~500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6,810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching. Jurkat T-cell mRNA was analyzed on an Illumina HiSeq2000. ~80 million paired end reads (2x200bp, ~350bp lengths) were collected.
2013-05-17 | E-GEOD-45428 | biostudies-arrayexpress
Project description:Many new alternative splice forms have been detected at the transcript level using next generation sequencing (NGS) methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of NGS, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splice-junction peptides. Results: Eighty million paired-end Illumina reads and ~500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6,810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching.
2013-05-17 | GSE45428 | GEO
Project description:The Muscleblind-like (Mbnl) family of RNA-binding proteins plays important roles in muscle and eye development and in Myotonic Dystrophy (DM), where expanded CUG or CCUG repeats functionally deplete Mbnl proteins. We identified transcriptome-wide functional and biophysical targets of Mbnl proteins in brain, heart, muscle, and myoblasts using RNA sequencing and crosslinking/immunoprecipitation-sequencing approaches. This analysis identified several hundred splicing events whose regulation depended on Mbnl function, in a pattern indicative of functional interchangeability between Mbnl1 and Mbnl2. A nucleotide resolution RNA map associated repression or activation of exon splicing with Mbnl binding near either 3' splice site or near the downstream 5' splice site, respectively. Transcriptomic analysis of sub-cellular compartments uncovered a global role for Mbnls in regulating localization of mRNAs encoding membrane, synaptic and other proteins in both mouse and Drosophila cells, and Mbnls also contribute to protein secretion. These findings hold several new implications for DM pathogenesis. To assess global functions of Muscleblind proteins, RNA-Seq was performed using WT and Mbnl1 KO brain, heart, and muscle (5 mice each). Additionally, C2C12 mouse myoblasts were depleted of Mbnl1, Mbnl2, or both. Subcellular fractionation experiments were performed to analyze mRNA localization following depletion of Mbnl1 and Mbnl2 in C2C12 mouse myoblasts, and following depletion of Mbnl in Drosophila S-2R+ cells. CLIP-Seq was also performed against Mbnl1 in mouse brain, heart, muscle, and C2C12 myoblasts. Finally, ribosome footprinting was performed with C2C12 mouse myoblasts that were depleted of Mbnl1, Mbnl2, or both.
2012-08-17 | E-GEOD-39911 | biostudies-arrayexpress
Project description:The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs requires internal ribosome entry sites (IRES) if they are to undergo translation without 5’ cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify over 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue -specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is down regulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease.
2021-08-25 | GSE178718 | GEO