RNA G-quadruplex (rG4) secondary structures are proposed to play key roles in fundamental biological processes that include the modulation of transcriptional, co-transcriptional, and ...posttranscriptional events. Recent methodological developments that include predictive algorithms and structure-based sequencing have enabled the detection and mapping of rG4 structures on a transcriptome-wide scale at high sensitivity and resolution. The data generated by these studies provide valuable insights into the potentially diverse roles of rG4s in biology and open up a number of mechanistic hypotheses. Herein we highlight these methodologies and discuss the associated findings in relation to rG4-related biological mechanisms.
The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and ...histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.
G-quadruplexes (G4s) are nucleic acid secondary structures that form within guanine-rich DNA or RNA sequences. G4 formation can affect chromatin architecture and gene regulation and has been ...associated with genomic instability, genetic diseases and cancer progression. Here we present a high-resolution sequencing-based method to detect G4s in the human genome. We identified 716,310 distinct G4 structures, 451,646 of which were not predicted by computational methods. These included previously uncharacterized noncanonical long loop and bulged structures. We observed a high G4 density in functional regions, such as 5' untranslated regions and splicing sites, as well as in genes previously not predicted to contain these structures (such as BRCA2). G4 formation was significantly associated with oncogenes, tumor suppressors and somatic copy number alterations related to cancer development. The G4s identified in this study may therefore represent promising targets for cancer intervention.
RNA secondary structures in the 5'-untranslated regions (5'-UTR) of mRNAs are key to the post-transcriptional regulation of gene expression. While it is evident that non-canonical Hoogsteen-paired ...G-quadruplex (rG4) structures somehow contribute to the regulation of translation initiation, the nature and extent of human mRNAs that are regulated by rG4s is not known. Here, we provide new insights into a mechanism by which rG4 formation modulates translation.
Using transcriptome-wide ribosome profiling, we identify rG4-driven mRNAs in HeLa cells and reveal that rG4s in the 5'-UTRs of inefficiently translated mRNAs associate with high ribosome density and the translation of repressive upstream open reading frames (uORF). We demonstrate that depletion of the rG4-unwinding helicases DHX36 and DHX9 promotes translation of rG4-associated uORFs while reducing the translation of coding regions for transcripts that comprise proto-oncogenes, transcription factors and epigenetic regulators. Transcriptome-wide identification of DHX9 binding sites shows that reduced translation is mediated through direct physical interaction between the helicase and its rG4 substrate.
This study identifies human mRNAs whose translation efficiency is modulated by the DHX36- and DHX9-dependent folding/unfolding of rG4s within their 5'-UTRs. We reveal a previously unknown mechanism for translation regulation in which unresolved rG4s within 5'-UTRs promote 80S ribosome formation on upstream start codons, causing inhibition of translation of the downstream main open reading frames. Our findings suggest that the interaction of helicases with rG4s could be targeted for future therapeutic intervention.
Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. ...It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.
Abstract
RNA G-quadruplexes (rG4s) are secondary structures in mRNAs known to influence RNA post-transcriptional mechanisms thereby impacting neurodegenerative disease and cancer. A detailed ...knowledge of rG4-protein interactions is vital to understand rG4 function. Herein, we describe a systematic affinity proteomics approach that identified 80 high-confidence interactors that assemble on the rG4 located in the 5′-untranslated region (UTR) of the NRAS oncogene. Novel rG4 interactors included DDX3X, DDX5, DDX17, GRSF1 and NSUN5. The majority of identified proteins contained a glycine-arginine (GAR) domain and notably GAR-domain mutation in DDX3X and DDX17 abrogated rG4 binding. Identification of DDX3X targets by transcriptome-wide individual-nucleotide resolution UV-crosslinking and affinity enrichment (iCLAE) revealed a striking association with 5′-UTR rG4-containing transcripts which was reduced upon GAR-domain mutation. Our work highlights hitherto unrecognized features of rG4 structure-protein interactions that highlight new roles of rG4 structures in mRNA post-transcriptional control.
Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, ...intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.
We describe a sequence-based computational model to predict DNA G-quadruplex (G4) formation. The model was developed using large-scale machine learning from an extensive experimental G4-formation ...dataset, recently obtained for the human genome via G4-seq methodology. Our model differentiates many widely accepted putative quadruplex sequences that do not actually form stable genomic G4 structures, correctly assessing the G4 folding potential of over 700,000 such sequences in the human genome. Moreover, our approach reveals the relative importance of sequence-based features coming from both within the G4 motifs and their flanking regions. The developed model can be applied to any DNA sequence or genome to characterise sequence-driven intramolecular G4 formation propensities.
Head and neck squamous cell carcinoma (HNSCC) remain a substantial burden to global health. Cell-free circulating tumour DNA (ctDNA) is an emerging biomarker but has not been studied sufficiently in ...HNSCC.
We conducted a single-centre prospective cohort study to investigate ctDNA in patients with p16-negative HNSCC who received curative-intent primary surgical treatment. Whole-exome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumour tissue. We utilised RaDaR
, a highly sensitive personalised assay using deep sequencing for tumour-specific variants, to analyse serial pre- and post-operative plasma samples for evidence of minimal residual disease and recurrence.
In 17 patients analysed, personalised panels were designed to detect 34 to 52 somatic variants. Data show ctDNA detection in baseline samples taken prior to surgery in 17 of 17 patients. In post-surgery samples, ctDNA could be detected at levels as low as 0.0006% variant allele frequency. In all cases with clinical recurrence to date, ctDNA was detected prior to progression, with lead times ranging from 108 to 253 days.
This study illustrates the potential of ctDNA as a biomarker for detecting minimal residual disease and recurrence in HNSCC and demonstrates the feasibility of personalised ctDNA assays for the detection of disease prior to clinical recurrence.
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