Biology subjects

Di Antonio, M.

Publications and source records attributed to Di Antonio, M..

4 recordsLinked to original sources

bioRxiv2023

Cellular visualization of G-quadruplex RNA via fluorescence lifetime imaging microscopy

Over the last decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance have rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro, however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescence probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independently of local probe concentration. Furthermore, TOR-G4 primarily co-localizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/539224v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@95c5faorg.highwire.dtl.DTLVardef@f8a897org.highwire.dtl.DTLVardef@1047f23org.highwire.dtl.DTLVardef@36e9ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

The C9Orf72 hexanucleotide repeat expansion transcript forms insoluble aggregates facilitated by multimolecular G-quadruplex structures

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is the expansion of the intronic hexanucleotide repeat (GGGGCC)nin C9orf72. Here, we investigated the formation of nucleic-acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of the diseases. We observed significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associated to the formation of multimolecular G-quadruplexes (mG4s), using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions led to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validated the biological relevance of our findings by demonstrating the ability of a G4-selective fluorescent probe to penetrate C9orf72 mutant human motor neurons derived from ALS patients, which revealed clear fluorescent signal in putative condensates. Our findings strongly suggest that RNA G- rich repetitive sequences can form protein-free condensates sustained by multimolecular G- quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation related ALS and FTD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/526399v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@18c11e6org.highwire.dtl.DTLVardef@eb308eorg.highwire.dtl.DTLVardef@c0c870org.highwire.dtl.DTLVardef@a7555a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

The Response of the Eukaryotic Replisome to G-quadruplex- and i-motif-forming Sequences

Sequences that can form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play a range of physiological roles. However, they can also pose a challenge to the replication machinery and in turn threaten genome stability. Multiple lines of evidence suggest G4s interfere with replication, but the underlying mechanism remains unclear. Moreover, there is a lack of evidence of how iMs affect the replisome. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM is sufficient to arrest DNA replication. Direct single molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. A combination of genetic and biophysical characterisation establishes that structure forming capacity is a key determinant of replisome arrest. Mechanistically, replication fork arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Sgs1 or Chl1. Altogether, this study provides a potential mechanism for quadruplex structure formation and resolution during replication and highlights G4s and iMs as endogenous sources of replication stress, which may explain their genomic instability and mutation frequencies in cancer.

molecular biology↗

bioRxiv2021

Cockayne Syndrome B protein selectively interacts and resolves intermolecular DNA G-quadruplex structures

Guanine-rich DNA can fold into secondary structures known as G-quadruplexes (G4s). G4s can form from a single DNA-strand (intramolecular) or from multiple DNA-strands (intermolecular), but studies on their biological functions have been often limited to intramolecular G4s, owing to the low probability of intermolecular G4s to form within genomic DNA. Herein, we report that the endogenous protein Cockayne Syndrome B (CSB) binds with picomolar affinity to intermolecular G4s, whilst displaying negligible binding towards intramolecular structures. We also observed that CSB can selectively resolve intermolecular G4s in an ATP independent fashion. Our study demonstrates that intermolecular G4s formed within ribosomal DNA are natural substrates for CSB, strongly suggesting that these structures might be formed in the nucleolus of living cells. Given that CSB loss of function elicits premature ageing phenotypes, our findings indicate that the interaction between CSB and ribosomal DNA intermolecular G4s is essential to maintain cellular homeostasis.

biochemistry↗