Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Analysis of lentiviral vector splicing events with nanopore sequencing

ABSTRACT: All lentiviral vectors derived from HIV-1 have the major splice donor (SD1) in the 5’ leader region and splice acceptor 7 (SA7) within the env region. Splicing events decrease the amount of full-length RNA available for packaging into virions and could lead to packaging of genomes containing internal deletions. Because there are splice sites or splicing enhancer/silencer elements in both gag and env, we compared how deleting each region affected intracellular genomic RNA splicing in RNA isolated from transfected HEK293Tsa cells. Oxford Nanopore direct cDNA sequencing was used to characterize the splice variants because the long-read length allows full-length transcripts to be analyzed. We show that deleting 507 nt of env, including the SA7, decreased the number of splicing events per transcript and increased the proportion of unspliced genomic RNAs ~3-fold in the cell.

ORGANISM(S): Homo sapiens

PROVIDER: GSE165829 | GEO | 2021/01/30

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

An epitranscriptomic switch at the 5´-UTR controls genome selection during HIV-1 genomic RNA packaging

Project description:During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA (gRNA). While the simple retrovirus MLV segregates its full-length RNA into two functional populations, the HIV-1 full-length RNA was proposed to exist as a single population used indistinctly for protein synthesis or packaging. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that an epitranscriptomic switch involving the demethylation of adenosine residues present within HIV-1 5´-UTR regulates full-length RNA packaging. We further identified two conserved adenosines within the 5’-UTR that have a crucial structural and functional role and that are modulated by N6-methylation. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promotes the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus contributing to full-length RNA demethylation. Finally, the specific inhibition of the FTO RNA demethylase activity suppressed HIV-1 full-length RNA packaging. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the full-length RNA molecules that will be used as viral genomes.

2021-01-02 | GSE130687 | GEO

New strategies for the identification of intronic variants related to splicing events in pancreas cancer [iCELL8]

Project description:Most clinical diagnostic settings and genomic research focus almost exclusively on coding regions and essential splice sites, mostly ignoring non-coding variants. Indeed, the investigation of intronic mis-splicing variants interpreting mechanisms to disease-associated splicing events requires both genomic and transcriptomic data. Unfortunately, there are not many datasets where both are available, leading to the understanding of intronic variants in diseases full of gaps. In this study, we present for the first time a full-length single nuclei RNA-sequencing (snRNA-Seq) approach improving the proper investigation of pathogenic mis-splicing intronic variants in pancreatic cancer showing its contribution to abnormal splicing changes and their transcriptional effects. Finally, we discuss the demands of further machine learning-based methods to process the growing number of single cell data and to enhance the precision and recall.

2024-05-10 | GSE229006 | GEO

New strategies for the identification of intronic variants related to splicing events in pancreas cancer [RNA-Seq]

2024-05-08 | GSE228844 | GEO

Effect of SF3A1 inhibition on pre-mRNA splicing

Project description:We previously found that the SF3A mRNA splicing complex was required for a robust innate immune response; SF3A acts in part by inhibiting the production of a negatively acting splice form of the TLR signaling adaptor MyD88. Here we inhibit SF3A1 using RNAi and subsequently perform an RNAseq study to identify the full complement of genes and splicing events regulated by SF3A in murine macrophages. Surprisingly, SF3A has substantial specificity for mRNA splicing events in innate immune signaling pathways compared to other pathways, affecting the splicing of many genes in the TLR signaling pathway to modulate the innate immune response.

2015-02-09 | GSE58432 | GEO

PRMT5 and CDK4/6 inhibition result in distinctive patterns of alternative splicing

Project description:To investigate the full effects of inhibition of CDK4/6 on splicing events in melanoma and the extent to which they are dependent on PRMT5 . We performed full-length mRNA sequencing on CHL1 and A375 melanoma cell lines treated with the CDK4/6 inhibitor palbociclib and the PRMT5 inhibitor GSK3326595 and CTX085 and analysed data for differential gene expression and differential pre-mRNA splicing induced by these agents.

2022-07-28 | GSE209753 | GEO

Insights into transcriptome surveillance and alternative splicing regulation by the EJC-associated splicing factor RNPS1

Project description:Alternative splicing of pre-mRNAs increases the potential for regulation and complexity of gene expression. The exon junction complex (EJC) and its associated splicing factor RNPS1 were recently shown to suppress mis-splicing resulting from the usage of cryptic and reconstituted 5’ and 3’ splice sites in the vicinity of the EJC. Here, we aimed to further investigate the mechanisms underlying splicing regulation by RNPS1. A transcriptome-wide analysis identified hundreds of splice events affected by the knockdown (KD) of RNPS1 in HeLa cells. These included alternative splice site usage as well as intron retention, exon skipping and inclusion. However, only a fraction of these RNPS1-dependent splice events was fully or partially rescued by the expression of the RNPS1 RRM. These results indicated that another domain of RNPS1 is involved in the regulation of the majority of splicing events. Deletion experiments revealed that the N-terminus and S-domain, and in particular the C-terminus of RNPS1 strongly regulate these events. Several splicing factors, including SR proteins and U1 snRNP components, were strongly reduced in the interactome of RNPS1 lacking the C terminus. We conclude that RNPS1 interacts with many splicing factors to direct the assembly of EJC-dependent and-independent splicing complexes.

2022-05-31 | PXD027251 | Pride

Whole genome analysis of cells permissive for late gene expression of HPV-16

Project description:Aim was to identify cellular factors that regulate HPV-16 late gene expression at the level of RNA processing Cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5‚Äô-splice site SD3632, produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c) and HuR that are known to regulate HPV16 late gene expression, or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the hnRNP C-family. Overexpression of RALYL, or RALY and hnRNP C1 that are two other members of the hnRNP C-family, induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. Induction of HPV16 late gene expression by the hnRNP C-proteins was dependent on the HPV16 early untranslated region to which these proteins also were shown to bind in vitro, and in living cells. Our experiments revealed that hnRNP C proteins that interacted with the HPV16 early untranslated region reached out to the splicing silencer complex at HPV16 SD3632 and derepressed this splice site, thereby activating production of HPV16 spliced late L1 mRNAs. In conclusion, hnRNP C- proteins bind to the HPV16 early untranslated region and control of HPV16 late L1 mRNA splicing. Total cellular RNA was extracted from stable cell lines. Samples were prepared in triplicates. C33ARSVNeo served as control cell line.

2015-06-12 | E-GEOD-65166 | biostudies-arrayexpress

Ultra-deep sequencing reveals pre-mRNA splicing as a sequence driven high-fidelity process

Project description:Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5’ splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5’ splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing.

2019-09-24 | GSE137883 | GEO

Transcriptomic analysis of testis and epididymis tissues from Banna mini-pig inbred line (BMI) boars with single-molecule long-read sequencing

Project description:The testis and epididymis tissues collected from 12-month-old adult BMI boars were carried out PacBio Iso-Seq sequencing and Illumina RNA-seq sequencing. The full-length isoforms, extensive alternative splicing events, lncRNAs, some genes and novel isoforms related to spermatogenesis were evaluated.

2022-12-10 | GSE193976 | GEO

Transcriptome of ΔPfPRMT5 parasite during malaria parasite asexual development [PRMT5_RNAseq]

Project description:Purpose: this study is to analyze the change of RNA splicing events after disruption of Protein Arginine Methyltranferase 5 (PRMT5) in Plasmodium falciparum. Methods: In this study, the transcriptomes of a PfPRMT5 gene knockout (KO) parasite line with its wildtype control were analyzed by RNAseq.Total RNA were harvested from the asexual parasites at four stages (ring, early trophozoite, late trophozoite, and schizont)(12h, 24h, 36h, and 46h post-invasion) using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Only reads that had a minimum length of 50 base pairs were retained. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Results: This RNAseq analysis with strand-specific mRNA libraries from both ΔPfPRMT5 and WT lines showed that >90% of sequencing reads were of high quality for mapping to the P. falciparum genome with nearly 40 times of coverage. This allows us to analyze the change of alternative splicing events (alternative 5' splice site, alternative 3' splice site, retained intron and skipped exon). 800, 1056, 479, and 1158 alternative splicing events (alternative 5' splice site, alternative 3' splice site, retained intron and skipped exon) were altered in the DPfPRMT5 parasite line as compared to the WT line at four development stages, respectively (unpublished data). Conclusions: Collectively, this RNAseq provide a dataset for analysis of abnormal RNA splicing events in the ΔPfPRMT5 parasites.

2023-06-28 | GSE199366 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data