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Transcriptome-wide RNA and genome-wide DNA G-Quadruplex mediates alternative transcripts

ABSTRACT: Transcriptome-wide RNA and genome-wide DNA G-Quadruplex mediates alternative transcripts

PROVIDER: PRJNA478594 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7456880_1.fastq.gz	Fastqsanger.gz
	SRR7456880_2.fastq.gz	Fastqsanger.gz
	SRR7456881_1.fastq.gz	Fastqsanger.gz
	SRR7456881_2.fastq.gz	Fastqsanger.gz
	SRR7456882_1.fastq.gz	Fastqsanger.gz

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Transcriptome-wide RNA and genome-wide DNA G-Quadruplex mediates alternative transcripts

Project description:We demonstrate that G4-DNA and G4-RNA may present splicing sites and alternative transcription starting positions on a transcriptome-wide and genome-wide scale by showing widespread effect on alternative transcripts in response to the established intracellular G4 binding ligands PDS.

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G-Quadruplex structures are stable and detectable in human genomic DNA.

Project description:The G-quadruplex is an alternative DNA structural motif that is considered to be functionally important in the mammalian genome. Herein, we address the hypothesis that G-quadruplex structures can exist within double-stranded genomic DNA using a G-quadruplex-specific probe. An engineered antibody is employed to enrich for DNA containing G-quadruplex structures, followed by deep sequencing to detect and map G-quadruplexes at high resolution in genomic DNA from human breast adenocarcinoma cells. Our high sensitivity structure-based pull-down strategy enables the isolation of genomic DNA fragments bearing a single as well as multiple G-quadruplex structures. Stable G-quadruplex structures are found in sub-telomeres, gene bodies and gene regulatory regions. For a sample of identified target genes, we show that G-quadruplex stabilizing ligands can modulate transcription. These results confirm the existence of G-quadruplex structures and their persistence in human genomic DNA. Four independent libraries have been enriched in DNA G-quadruplex structures using a G-quadruplex-specific probe. One genomic input library was sequenced as control. Deep-sequencing of these libraries allowed the mapping of G-quadruplexes on the genome.

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RNA G-quadruplex secondary structure promotes alternative splicing via the RNA binding protein hnRNPF

Project description:It is generally thought that splicing factors regulate alternative splicing through binding to RNA consensus sequences. In addition to these linear motifs, RNA secondary structure is emerging as an important layer in splicing regulation. Here we demonstrate that RNA elements with G-quadruplex forming capacity promote exon inclusion. Destroying G-quadruplex forming capacity while keeping G-tracts intact abrogates exon inclusion. Analysis of RNA binding protein footprints revealed that G-quadruplexes are enriched in hnRNPF-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome. Moreover, hnRNPF regulates an EMT-associated CD44 isoform switch in a G-quadruplex-dependent manner, which results in inhibition of EMT. Mining breast cancer TCGA datasets, we demonstrate that hnRNPF negatively correlates with an EMT gene signature and positively correlates with patient survival. These data suggest a critical role for RNA G-quadruplexes in regulating alternative splicing. Modulation of G-quadruplex structural integrity may control cellular processes important for tumor progression.

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Systematic identification of G-quadruplex sensitive lethality by genome-wide genetic screening

Project description:A375 and HT1080 cells are treated with G-quadruplex ligands PDS or PhenDC3 following genome-wide shRNA knockdown. This enables the identification of genes that when silenced, specifically compromises cell growth in the presence of the ligand. First, a pilot screen was performed to determine a ligand concentration and experimental duration that caused ligand-specific, significant changes in shRNA levels. Second, a genome-wide screen was performed to globally evaluate G4-ligand synthetic lethal interactions. Third, to corroborate the G4-sensitisers uncovered in the genome-wide screen, a focussed screen was performed with a custom shRNA pool.

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