Detecting intragenic trans-splicing events with hybrid transcriptome sequencing in cancer cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE233510 | GEO | 2023/05/25
REPOSITORIES: GEO
Similar Datasets
Project description:Trans-spliced RNAs (ts-RNAs) are a type of non-co-linear (NCL) transcripts that consist of exons in an order topologically inconsistent with the corresponding DNA template. Detecting ts-RNAs is often interfered by experimental artifacts, circular RNAs (circRNAs) and genetic rearrangements. Particularly, intragenic ts-RNAs, which are derived from separate precursor mRNA molecules of the same gene, are often mistaken for circRNAs through analyses of RNA-seq data. Here we developed a bioinformatics pipeline (NCLscan-hybrid), which integrated short and long RNA-seq reads to minimize false positives and proposed out-of-circle and rolling-circle long reads to distinguish between intragenic ts-RNAs and circRNAs. Combining NCLscan-hybrid screening and multiple experimental validation steps successfully confirmed that four NCL events, which were previously regarded as circRNAs in databases, originated from trans-splicing. CRISPR-based endogenous genome modification experiments further showed that flanking intronic complementary sequences can significantly contribute to ts-RNA formation, providing an efficient/specific method to deplete ts-RNAs. We also experimentally validated that one ts-RNA (ts-ARFGEF1) played an important role for p53-mediated apoptosis through affecting the PERK/eIF2a/ATF4/CHOP signaling pathway in breast cancer cells. This study thus described both bioinformatics procedures and experimental validation steps for rigorous characterization of ts-RNAs, expanding future studies for identification, biogenesis, and function of these important but understudied transcripts.
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Project description:Detecting intragenic trans-splicing events with hybrid transcriptome sequencing in cancer cells
| PRJNA976465 | ENA
Project description:Numerous studies have described the altered expression and the causal role of miRNAs in human cancer. However, to date efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here, we find that Nucleolin (NCL), a major nucleolar protein, post-transcriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, causally involved in breast cancer initiation, progression and drug-resistance. We also show that NCL is commonly overexpressed in human breast tumors, and its expression correlates with that of NCL-dependent miRNAs. Finally, this study indicates that NCL-binding guanosine-rich aptamers affect the levels of NCL-dependent miRNAs and their target genes, reducing breast cancer cell aggressiveness, both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer. Identification of NCL regulated miRNAs by using miRNA high-throughput sequencing of HeLa cells stably expressing double-strand (ds) interfering RNA against NCL or scrambled sequences (sh-NCL or sh-Scr).
2013-02-07 | E-GEOD-44052 | biostudies-arrayexpress
Project description:To explore the landscape of intergenic trans-splicing events and characterize their functions and evolutionary dynamics, we conduct a mega-data study of a phylogeny containing eight species across five orders of class Insecta, a model system spanning 400 million years of evolution. A total of 1,627 trans-splicing events involving 2,199 genes are identified, accounting for 1.58% of the total genes. Homology analysis reveals that mod(mdg4)-like trans-splicing is the only conserved event that is consistently observed in multiple species across two orders, which represents a unique case of functional diversification involving trans-splicing. Thus, evolutionarily its potential for generating proteins with novel function is not broadly utilized by insects. Furthermore, 146 non-mod trans-spliced transcripts are found to resemble canonical genes from different species. Trans-splicing preserving the function of 'breakup' genes may serve as a general mechanism for relaxing the constraints on gene structure, with profound implications for the evolution of genes and genomes.
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Project description:Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5'-gene with 46 newly identified alternative 3'-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F(1) hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies.
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Project description:The poly A binding protein RB47 was purified from C. reinhardtii chloroplasts and was found to have endoribonuclease activity. MS analysis suggested that an internal non-conserved linker (NCL)sequence was absent from the native protein. Recombinant, full length RB47 protein, including the NCL did not have endoribonuclease activity while versions lacking the NCL were active. An in vitro system, utilizing recombinant RB47 and chloroplast lysates, recreated the processing of RB47 in that the NCL was excised and the flanking sequences were spliced together.
2018-10-27 | PXD004285 | Pride
Project description:Trans-spliced isoform of NCRMS (tsNCRMS) tends to be specifically transcribed in hESCs/iPSCs and disruption of tsNCRMS expression can conspicuously affect the pluripotency maintenance of hESCs. We disrupt the expression of tsNCRMS by introducing tsNCRMS shTS2 that target the no-co-linear junction site in hESCs using lentiviral vectors and use shLuc as control.
2013-09-30 | E-GEOD-32503 | biostudies-arrayexpress
Project description:Numerous studies have described the altered expression and the causal role of miRNAs in human cancer. However, to date efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here, we find that Nucleolin (NCL), a major nucleolar protein, post-transcriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, causally involved in breast cancer initiation, progression and drug-resistance. We also show that NCL is commonly overexpressed in human breast tumors, and its expression correlates with that of NCL-dependent miRNAs. Finally, this study indicates that NCL-binding guanosine-rich aptamers affect the levels of NCL-dependent miRNAs and their target genes, reducing breast cancer cell aggressiveness, both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer. HeLa cells were transfected with the control or anti-nucleolin siRNA. After 72hours total RNA was collected and analyzed by NanoString.
2012-11-02 | E-GEOD-41971 | biostudies-arrayexpress
Project description:Numerous studies have described the altered expression and the causal role of miRNAs in human cancer. However, to date efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here, we find that Nucleolin (NCL), a major nucleolar protein, post-transcriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, causally involved in breast cancer initiation, progression and drug-resistance. We also show that NCL is commonly overexpressed in human breast tumors, and its expression correlates with that of NCL-dependent miRNAs. Finally, this study indicates that NCL-binding guanosine-rich aptamers affect the levels of NCL-dependent miRNAs and their target genes, reducing breast cancer cell aggressiveness, both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer. MCF7 cells were treated with the control drug or AS1411 aptamer. After 72hours total RNA was collected and analyzed by Affymetrix U133 plus.
2012-11-02 | E-GEOD-41972 | biostudies-arrayexpress
Project description:Trans-splicing is a post-transcriptional event that joins exons from separate pre-mRNAs. Detection of trans-splicing is usually severely hampered by experimental artifacts and genetic rearrangements. Here, we develop a new computational pipeline, TSscan, which integrates different types of high-throughput long-/short-read transcriptome sequencing of different human embryonic stem cell (hESC) lines to effectively minimize false positives while detecting trans-splicing. Combining TSscan screening with multiple experimental validation steps revealed that most chimeric RNA products were platform-dependent experimental artifacts of RNA sequencing. We successfully identified and confirmed four trans-spliced RNAs, including the first reported trans-spliced large intergenic noncoding RNA ("tsRMST"). We showed that these trans-spliced RNAs were all highly expressed in human pluripotent stem cells and differentially expressed during hESC differentiation. Our results further indicated that tsRMST can contribute to pluripotency maintenance of hESCs by suppressing lineage-specific gene expression through the recruitment of NANOG and the PRC2 complex factor, SUZ12. Taken together, our findings provide important insights into the role of trans-splicing in pluripotency maintenance of hESCs and help to facilitate future studies into trans-splicing, opening up this important but understudied class of post-transcriptional events for comprehensive characterization
2013-09-30 | GSE32503 | GEO