RNA G-quadruplex secondary structure promotes alternative splicing via the RNA binding protein hnRNPF
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ABSTRACT:
PROVIDER: PRJNA420499 | ENA |
REPOSITORIES: ENA
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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.
2018-01-22 | GSE107542 | GEO
Project description: Not available
| S-EPMC5769772 | biostudies-literature
Project description:RNA secondary structures have been increasingly reported to serve critical regulatory roles in post-transcriptional gene regulation. RNA G-quadruplex secondary structures can serve as cis-elements to recruit splicing factors and regulate alternative RNA splicing. We recently showed that RNA G-quadruplexes play a critical regulatory role in regulating alternative splicing during the epithelial mesenchymal transition. Due to the critical role alternative splicing plays in human health and disease, an unmet need exists to identify small molecule modulators of alternative splicing. In this study, we performed high-throughput screening using a dual-output splicing reporter to identify small molecules capable of regulating alternative splicing by interacting with RNA secondary structure G-quadruplexes. We identify emetine and its analog cephaeline as small molecules that denature RNA G-quadruplexes in a sequence and location independent manner to modify alternative splicing. Transcriptome analysis reveals that treatment with emetine globally regulates alternative splicing, including events associated with exon-proximal G-quadruplexes. These data suggest a critical role for emetine and cephealine as splicing regulators with the selective ability to disrupt RNA G-quadruplex-associated alternative splicing in vivo.
2019-02-01 | GSE113505 | GEO
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.
2013-05-08 | E-GEOD-45241 | biostudies-arrayexpress
Project description:Drak2 Regulates RNA Alternative Splicing by Suppressing SRPK1-SRSF6 Signaling Pathway
2021-09-17 | PXD028609 | iProX
Project description:MALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNA-G-quadruplexes (rG4s) present in the 3’ region of MALAT1 which regulates this function. Using rG4 domain specific RNA-pull-down followed by mass-spectrometry, RNA-immuno-precipitation and imaging, we demonstrate the rG4 dependent localization of Nucleolin (NCL) and Nucleophosmin (NPM) to nuclear speckles. Specific G-to-A mutations that abolish rG4 structures, results in the localization loss of both the proteins from speckles. Functionally, disruption of rG4 in MALAT1 phenocopies NCL knockdown resulting in altered pre-mRNA splicing of endogenous genes. These results reveal a central role of rG4s within the 3’ region of MALAT1 orchestrating AS.
2022-02-17 | PXD026386 | Pride
Project description:G-quadruplex RNA pulldown followed by mass spectrometry. G4A4 oligoribonucleotide (5’-AAAAAAGGGGAAAAGGGGAAAAGGGGAAAAGGGGAAAAAA-3’) was biotinylated and folded in either K+ or Li+ containing buffers, causing folding of G-quadruplex (K+) or destabilization of the secondary structure (Li+). G4A4 pulldown was used to identify G4-RNA binding proteins from K562 human cell line nuclear extracts, interacting in structure-dependent manner.
2024-02-20 | PXD041154 | Pride
Project description:Post-transcriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure, and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of RNAs in the nucleus has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP binding site mapping approach on Arabidopsis seedling nuclei in vivo to globally profile these features within the nuclear compartment. We reveal opposing patterns of secondary structure and RBP binding levels throughout native messenger RNAs that demarcate alternative splicing and polyadenylation. We also uncover a collection of protein bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, we identify a nuclear role for the chloroplast RBP, CP29A. In total, we provide the first simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus. Protein interaction profile sequencing (PIP-seq) in Arabidopsis seedling nuclei. These are crosslinked with formaldehyde and treated with two RNases (ssRNase and dsRNase) with two replicates
2014-12-16 | E-GEOD-58974 | biostudies-arrayexpress
Project description:G-quadruplex structures in the 5’ UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry. We found different patterns of interactions for the G-quadruplex motifs under investigation. While the G-quadruplexes in the mRNAs of NRAS and MMP16 specifically interacted with a small number of proteins, the Bcl-2 and ARPC2 G-quadruplexes exhibited a broad range of proteinaceous interaction partners with 99 and 82 candidate proteins identified in at least two replicates, respectively. Among the interaction partners were many proteins known to bind to RNA, including multiple heterogenous nuclear ribonucleoproteins (hnRNPs). The identified ribosomal proteins are likely to reflect stalling of the ribosome by RNA G-quadruplex structures. In addition, several proteins were identified that have not previously been described to interact with RNA. Gene ontology analysis of the set of candidate proteins revealed that many interaction partners are known to be tumor related. The majority of the identified RNA G-quadruplex interacting proteins are thought to be involved in post-transcriptional processes, particularly in splicing. These findings indicate that protein-G-quadruplex interactions may be relevant to the regulation of mRNA maturation and may play an important role in tumor biology.
2017-11-14 | PXD005761 | Pride
Project description:RNA-binding proteins are important regulators of RNA biogenesis and function, and recent work shows that chromatin- and RNA-binding proteins can have regulatory roles in transcription. Several approaches have been developed to predict RNA-binding proteins and to identify them experimentally at large scale, including binding to structural elements as G-quadruplexes. Here, we show that the histone lysine demethylase protein KDM5 can bind to RNA through interaction with G-quadruplexes with high affinity despite neither being categorized as an RNA-binding protein nor identified as a G-quadruplex binding protein across numerous experimental large-scale and computational studies. In addition to characterizing the KDM5 G-quadruplex interaction we show that RNA is directly involved in the formation of KDM5-containing protein complexes. Computational predictions and comparison to other ARID domain containing proteins suggest that the ARID domain is directly interacting with both DNA and RNA across several proteins. Our work highlights that non-canonical aspects of RNA-binding proteins are present, and that G-quadruplex RNA-binding could provide a regulatory mechanism for chromatin-binding proteins with impact on transcription through linking protein complexes within the cell.
2024-04-11 | PXD050767 | Pride