POST-TRANSCRIPTIONAL REGULATION OF KRAS GENE: MECHANISM INVOLVING 5’UTR RNA G-QUADRUPLEXES, LONG NONCODING RNA AND NUCLEAR RIBONUCLEOPROTEIN A1
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE283011 | GEO | 2025/09/24
REPOSITORIES: GEO
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Project description:RNA structures are of crucial importance for biological function and gene regulation. Guanine (G)-rich sequences in RNA can assemble to form RNA G-quadruplex (rG4) structures; specific rG4s have been shown to regulate gene expression, and are associated with human diseases. However, no transcriptome-wide method has been reported to map rG4s, limiting our understanding of the rG4 structure and its relationship with function and regulation. Here we introduce a transcriptome-wide rG4 profiling method, rG4-Seq, in which the rG4-mediated reverse transcriptase stalling is read out by next-generation sequencing at nucleotide resolution. We apply this method to human RNA and report the first global map of rG4 structures for any organism. Our analysis identifies over 3,000 rG4s, and increases to over 11,000 upon addition of a rG4 stabilizing ligand Pyridostatin (PDS). Notably, we discover that besides canonical rG4s, many rG4s are non-canonical with novel structural features such as long-loops, bulges, and 2-quartet. We also find that rG4 formation is associated with its stability, Cytosine (C)-content, and the stability of alternative RNA structures. Our data reveals that rG4s can be found in messenger RNAs (mRNAs) and long intergenic noncoding RNAs (lincRNAs). In mRNAs, rG4s are enriched in untranslated regions, and are significantly correlated with microRNA target sites and polyadenylation signals. Remarkably, we found that majority of our in vitro identified rG4s can change the in silico predicted RNA structure to uncover alternative RNA conformation.. Futhermore, the rG4s that have analogues in many ortholog genes across different species show a preferential association with RNA processing and stability. rG4-seq is a broadly applicable method that enables the RNA G4 structurome and its biological roles to be interrogated and can be readily applied in other organisms on a transcriptome-wide scale. 12 samples, 150 bp single-ended RNA-Seq libraries from cell extracts after performing RTS reaction in different buffers (Li, K and K+PDS rich buffer). Each of the 3 conditions has 4 libraries from independent biological replicates.
2016-08-10 | E-GEOD-77282 | biostudies-arrayexpress
Project description:Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under permissive conditions. Although many RNAs contain sequences that fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. Here, we showed that the folding of putative rG4s in human cells into rG4 structures was dynamically induced by stress. By using high-throughput dimethylsulfate probing, we identified hundreds of endogenous stress-induced rG4s. Our results demonstrated that stress-induced rG4s were enriched in mRNA 3′-untranslated regions and enhanced mRNA stability. Furthermore, stress-induced rG4 folding was readily reversible upon stress removal. In summary, our study revealed the dynamic regulation of rG4 folding in living human cells and suggests suggested that widespread rG4 motifs may have a global regulatory impact on mRNA stability and cellular stress response.
2022-12-15 | GSE200706 | GEO
Project description:RNA structures are of crucial importance for biological function and gene regulation. Guanine (G)-rich sequences in RNA can assemble to form RNA G-quadruplex (rG4) structures; specific rG4s have been shown to regulate gene expression, and are associated with human diseases. However, no transcriptome-wide method has been reported to map rG4s, limiting our understanding of the rG4 structure and its relationship with function and regulation. Here we introduce a transcriptome-wide rG4 profiling method, rG4-Seq, in which the rG4-mediated reverse transcriptase stalling is read out by next-generation sequencing at nucleotide resolution. We apply this method to human RNA and report the first global map of rG4 structures for any organism. Our analysis identifies over 3,000 rG4s, and increases to over 11,000 upon addition of a rG4 stabilizing ligand Pyridostatin (PDS). Notably, we discover that besides canonical rG4s, many rG4s are non-canonical with novel structural features such as long-loops, bulges, and 2-quartet. We also find that rG4 formation is associated with its stability, Cytosine (C)-content, and the stability of alternative RNA structures. Our data reveals that rG4s can be found in messenger RNAs (mRNAs) and long intergenic noncoding RNAs (lincRNAs). In mRNAs, rG4s are enriched in untranslated regions, and are significantly correlated with microRNA target sites and polyadenylation signals. Remarkably, we found that majority of our in vitro identified rG4s can change the in silico predicted RNA structure to uncover alternative RNA conformation.. Futhermore, the rG4s that have analogues in many ortholog genes across different species show a preferential association with RNA processing and stability. rG4-seq is a broadly applicable method that enables the RNA G4 structurome and its biological roles to be interrogated and can be readily applied in other organisms on a transcriptome-wide scale.
2016-08-10 | GSE77282 | GEO
Project description:Following meiosis resumption, the transcription was silencing; and large number of maternal mRNA was actively translated and degraded during oocyte maturation. However, due to the limitation of sample number and technology, the dynamic distribution of RNA structure and its correlation with RNA dynamic change have never been revealed. RNA G-quadruplex (rG4) was a four-stranded RNA secondary structure related to translation regulation and RNA stability. In this study, we developed a low-input technique of rG4 detection (G4-lace-seq), and found that rG4s were highly abundant in fully grown oocytes and reduced during oocyte maturation. We ingeniously applied rG4 stable ligand BYBX in aim to eliminate the DNA G-quadruplex obstruction, and phenotypic analysis showed that rG4 accumulation severely impaired oocytes maturation. Above all, RBPs proteomic spectra revealed RNA structure change caused by G4 accumulation directly weakened the binding of RBPs to RNA, especially the enrichment of G4s affect the RBP binding associated with RNA metabolism, and G4s on the 5′-UTR hindered 40s ribosome movement and binding of 60s ribosome eukaryotic translation initiation factors to RNA. Combined analysis of G4-Lace-seq with Ribo-lite and RNA-seq, further proved that rG4s accumulation on UTRs disrupted translational efficiency and maternal mRNA degradation during oocyte maturation. Overexpression of DHX36, a G4s helicase, rescued the defects caused by BYBX to oocytes and restored the development rates. Jointly analyzing the function and distribution of rG4s and the metabolic signature of maternal mRNA, we hypothesized that a large amount of rG4s present before meiosis resumption is necessary to protect long-term mRNA stability from degradation in a state of poor efficiency translation. Following meiosis resumption, rG4s were cleaned up, translation efficiency increased, and the mRNA became unstable and was rapidly degraded. Collectively, our results announced for the first time that G4, as a secondary structure, had a hand in in the special dynamic rule of RNA content and translation. rG4s clearance determined the fate of cytoplasmic maturation during oocyte maturation.
2025-03-29 | GSE262943 | GEO
Project description:Following meiosis resumption, the transcription was silencing; and large number of maternal mRNA was actively translated and degraded during oocyte maturation. However, due to the limitation of sample number and technology, the dynamic distribution of RNA structure and its correlation with RNA dynamic change have never been revealed. RNA G-quadruplex (rG4) was a four-stranded RNA secondary structure related to translation regulation and RNA stability. In this study, we developed a low-input technique of rG4 detection (G4-lace-seq), and found that rG4s were highly abundant in fully grown oocytes and reduced during oocyte maturation. We ingeniously applied rG4 stable ligand BYBX in aim to eliminate the DNA G-quadruplex obstruction, and phenotypic analysis showed that rG4 accumulation severely impaired oocytes maturation. Above all, RBPs proteomic spectra revealed RNA structure change caused by G4 accumulation directly weakened the binding of RBPs to RNA, especially the enrichment of G4s affect the RBP binding associated with RNA metabolism, and G4s on the 5′-UTR hindered 40s ribosome movement and binding of 60s ribosome eukaryotic translation initiation factors to RNA. Combined analysis of G4-Lace-seq with Ribo-lite and RNA-seq, further proved that rG4s accumulation on UTRs disrupted translational efficiency and maternal mRNA degradation during oocyte maturation. Overexpression of DHX36, a G4s helicase, rescued the defects caused by BYBX to oocytes and restored the development rates. Jointly analyzing the function and distribution of rG4s and the metabolic signature of maternal mRNA, we hypothesized that a large amount of rG4s present before meiosis resumption is necessary to protect long-term mRNA stability from degradation in a state of poor efficiency translation. Following meiosis resumption, rG4s were cleaned up, translation efficiency increased, and the mRNA became unstable and was rapidly degraded. Collectively, our results announced for the first time that G4, as a secondary structure, had a hand in in the special dynamic rule of RNA content and translation. rG4s clearance determined the fate of cytoplasmic maturation during oocyte maturation.
2025-02-03 | GSE262730 | GEO
Project description:Following meiosis resumption, the transcription was silencing; and large number of maternal mRNA was actively translated and degraded during oocyte maturation. However, due to the limitation of sample number and technology, the dynamic distribution of RNA structure and its correlation with RNA dynamic change have never been revealed. RNA G-quadruplex (rG4) was a four-stranded RNA secondary structure related to translation regulation and RNA stability. In this study, we developed a low-input technique of rG4 detection (G4-lace-seq), and found that rG4s were highly abundant in fully grown oocytes and reduced during oocyte maturation. We ingeniously applied rG4 stable ligand BYBX in aim to eliminate the DNA G-quadruplex obstruction, and phenotypic analysis showed that rG4 accumulation severely impaired oocytes maturation. Above all, RBPs proteomic spectra revealed RNA structure change caused by G4 accumulation directly weakened the binding of RBPs to RNA, especially the enrichment of G4s affect the RBP binding associated with RNA metabolism, and G4s on the 5′-UTR hindered 40s ribosome movement and binding of 60s ribosome eukaryotic translation initiation factors to RNA. Combined analysis of G4-Lace-seq with Ribo-lite and RNA-seq, further proved that rG4s accumulation on UTRs disrupted translational efficiency and maternal mRNA degradation during oocyte maturation. Overexpression of DHX36, a G4s helicase, rescued the defects caused by BYBX to oocytes and restored the development rates. Jointly analyzing the function and distribution of rG4s and the metabolic signature of maternal mRNA, we hypothesized that a large amount of rG4s present before meiosis resumption is necessary to protect long-term mRNA stability from degradation in a state of poor efficiency translation. Following meiosis resumption, rG4s were cleaned up, translation efficiency increased, and the mRNA became unstable and was rapidly degraded. Collectively, our results announced for the first time that G4, as a secondary structure, had a hand in in the special dynamic rule of RNA content and translation. rG4s clearance determined the fate of cytoplasmic maturation during oocyte maturation.
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Project description:G-quadruplexes (rG4s) are recognized as key structures involved in RNA biology and are linked to neurodegenerative disease and cancer. However, detailed knowledge of rG4s and their binding partners is lacking. Here, a systematic, unbiased affinity proteomics approach identified 80 potential high-confidence interactors of the rG4 structure in the 5’UTR of the NRAS oncogene. Using epitope-tagged protein expression, we validated a subset of interactions including DDX3X, DDX5, DDX17, DHX9, DHX36, FXR1, FXR2, GRSF1 and NSUN5. More than half of the identified rG4 interactors were enriched in glycine-arginine (GAR) domains, and for DDX3X and DDX17, we show that the GAR domain is required for rG4 interaction. Transcriptome-wide binding analysis using DDX3X iCLIP revealed a striking association with rG4s in 5’UTRs that was lacking in the GAR-domain mutant. Overall, our work highlights a hitherto unrecognized complexity in rG4 structure-protein interactions that should be considered for the understanding of rG4 function.
2018-04-30 | GSE106476 | GEO
Project description:Translational control is a key determinant of protein abundance, which in turns defines the physiology and pathology of human cells. Initiation of translation is highly regulated in eukaryotes and is considered as the rate-limiting step of protein synthesis. mRNA secondary structures in 5’ untranslated region (UTR) and associated helicases have been characterised as key determinants of translation initiation. Nevertheless the transcriptome-wide contribution of non-canonical secondary structures, such as RNA G-quadruplexes (rG4s), to the translation of human mRNAs remains largely unappreciated. Here we use a ribosome profiling strategy to investigate the translational landscape associated to rG4s-containing mRNAs and the contribution of two rG4s-specialised DExH-box helicases, DHX9 and DHX36, to translation initiation in human cells. We show that rG4-forming sequences in 5’-UTR is associated with decreased translation efficiency which correlate with an increased ribosome density within the 5’-UTRs. We found that rG4s contribute to the translation of upstream open reading frames, and as a consequence, thwart the translation of the associated protein coding sequences (CDS). Depletion of the DHX36 and DHX9 helicases demonstrated that the formation of the rG4 structural motif rather than its nucleotide sequence mediate translation initiation. Our findings unveil a role for non-canonical structures in defining alternative 5’ starts for human mRNAs translation initiation.
2018-04-30 | GSE105082 | GEO
Project description:DDX21 is an RNA helicase protein with a diverse set of biological functions. Abnormal levels of DDX21 protein has been observed in colorectal and breast cancers as well as in schizophrenic patients, making it a potential therapeutic target. DDX21 can bind and unwind guanine-rich four-stranded structures of RNA referred to as guanine-quadruplexes (rG4s) and affect the translation of a reporter construct with an rG4 in its 5’ UTR. However, no biological rG4 targets of DDX1 are currently known. In our current study, we use shotgun proteomics to compare global protein expression of cells with wild type DDX21 and mutant DDX21 with impaired rG4s binding capability to identify potential binding targets of DDX21.
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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