Invited Speakers

DNA secondary structure and topology are in dynamic flux. Important question is what is the functional significance of non-canonical DNA structures. Sequences containing runs of guanines and capable of forming stable G4 DNA are highly enriched at gene promoters suggesting their potential role in regulating transcription. Recently, human Zn-finger transcription factors such as Sp1 and YY1 were shown to be G4 DNA-binding proteins. Our preliminary experiments led to the novel discovery that yeast transcription factor Msn2 binds to G4 DNA in vivo and in vitro. And G4 DNA forming sequences (PQS) are present at many of Msn2-target gene promoters. Transcription at these genes are up-regulated by G4 ligands. Here, we describe the kinetics and specificities of Msn2 interaction with G4 DNA and the effect of Msn2-G4 interaction in the regulation of transcription. We identified additional Zn-Finger transcription factors in both yeast and human that bind to G4 DNA with high affinity in support of a novel mode of transactivation in which G4 DNA becomes a docking site for multiple TFs including Msn2 to establish a hub of transactivation.

A common feature of most viruses is their dependence on regulatory RNA elements. Recent research suggested that non-canonical structures called G-quadruplexes (G4s) are overrepresented in viral genomes and have emerged as promising anti-viral targets. Using yellow fever virus (YFV) as a model system, we characterized the formation and the biological consequences of a conserved G4 within the genomes of the Flaviviridae family. We determined that this G4 is essential to promote viral replication and suppress the host cell stress response pathway. In subsequent mechanistic analyses, we pinpoint that this unique G4 function is associated with the nuclear host protein hnRNPH1. Specifically, G4 interaction leads to the retention of hnRNPH1 in the cytoplasm, causing an impaired stress response and alleviation of the anti-viral effects of stress-induced G4-forming tRNA fragments (tiRNAs). In conclusion, our data reveal a unique interplay of hnRNPH1 with host and viral G4 targets, controlling the integrated stress response and viral infection.

Synucleinopathies, including Parkinson’s disease, are triggered by alpha-synuclein aggregation, triggering progressive neurodegeneration. We demonstrated that RNA G-quadruplex assembly forms scaffolds for alpha-synuclein aggregation, contributing to neurodegeneration. Purified alpha-synuclein binds RNA G-quadruplexes directly through the N terminus. RNA G-quadruplexes undergo Ca2+-induced phase separation and assembly, accelerating α-synuclein sol-gel phase transition. In alpha-synuclein preformed fibril-treated neurons, RNA G-quadruplex assembly comprising synaptic mRNAs co-aggregates with alpha-synuclein upon excess cytoplasmic Ca2+ influx, eliciting synaptic dysfunction. Forced RNA G-quadruplex assembly using an optogenetic approach evokes α-synuclein aggregation, causing neuronal dysfunction and neurodegeneration. The administration of 5-aminolevulinic acid, a protoporphyrin IX prodrug, prevents RNA G-quadruplex phase separation, thereby attenuating α-synuclein aggregation in alpha-synuclein preformed fibril-injected synucleinopathic mice (Cell. 2024). In this symposium, I will also present the relevance of RNA G-quadruplex to other neurodegenerative diseases.

G-quadruplexes (G4s) can recruit transcription factors to activate gene expression, but detailed mechanisms are not well characterized. We demonstrate that G4s in the CCND1 promoter promote the molecular motility in MAZ phase-separated condensates and subsequently activate CCND1 transcription. Zinc finger (ZF) 2 of MAZ is responsible for G4 binding, while ZF3-5, but not a highly disordered region, is critical for MAZ condensation. MAZ nuclear puncta overlaps with signals of G4s and various coactivators including BRD4, MED1, CDK9 and active RNA polymerase II, as well as gene activation histone markers. MAZ mutants lacking either G4 binding or phase separation ability did not form nuclear puncta, and showed deficiencies in promoting hepatocellular carcinoma cell proliferation and xenograft tumor formation. Overall, we unveiled a novel mechanism that G4s recruit MAZ to the CCND1 promoter and facilitate the motility in MAZ condensates that compartmentalize coactivators to activate CCND1 expression and subsequently exacerbate hepatocarcinogenesis.

The pUG fold is an unusual left-handed (Z-form) quadruplex that forms in poly(UG)-rich RNA sequences.  We determined the high-resolution structure of the pUG fold and showed it is responsible for directing the amplification of RNA interference. We discovered that pUG folds mark mRNAs as vectors for the transgenerational epigenetic inheritance of gene silencing in vivo. We used several orthogonal methods, including atomic-level mutagenesis of reporter RNAs containing 7-deazaguanosine, to demonstrate this. The injection of atomically modified RNAs into live animals, accompanied by measurement of the silencing efficiency of GFP reporters, allowed us to provide direct evidence for functional importance of the pUG fold. I will describe the unusual biophysical properties of pUG RNAs, including their structure, dynamics and ability to form high-order multimeric interactions that enable efficient formation of condensates. I will also describe the “rules” that govern pUG folding and will show many sequences that are not poly(UG) can still adopt this unusual fold. These data lead to the identification of many thousands of potential pUG folds in regulatory regions of human RNAs, and some of these are associated with disease.  The rules for understanding pUG folding broaden our understanding of nucleic acid quadruplexes and will be important in the future for understanding when and where pUG folds form within transcriptomes.

Prion protein (PrP) causes prion diseases. PrP also functions as a receptor of Aβ protein to transmit the pathological signal of Aβ into cells, resulting in the repression of long-term potentiation (LTP) (Lauren et al., Nature, 2009). We isolated an RNA aptamer, that forms quadruplex, against the PrP, and determined the structure in complex with PrP. This clarified a mechanism how the aptamer exerts high affinity (Mashima et al., NAR, 2013). We also demonstrated spectroscopically that this aptamer inhibits the interaction between Aβ and its receptor, PrP, through tight binding to PrP (Iida et al., FEBS J., 2019). Recently, we have revealed electrophysiologically that the aptamer rescues the LTP repressed by Aβ, through the inhibition of the Aβ-receptor (PrP) interaction (Nakao, et al., in preparation).   We succeeded in observing the in-cell NMR signals of nucleic acids in living human cells for the first time. Then, we found that a G:G base pair of the quadruplex DNA opens more frequently in living cells than in vitro (Yamaoki et al., Nature Commun., 2022; Eladl et al., Chem. Commun., 2022; Eladl et al., Int. J. Mol. Sci., 2023; Eladl et al., submitted).

G-quadruplexes (GQs) are noncanonical DNA secondary structures composed of stacks of G-tetrads. They play important biological roles and have therapeutic potential as drug targets. Although many 2-4 G-tetrad GQs have been well characterized, monomolecular sequences with the potential to form 5 or more G-tetrads have not. Here, we investigate sequences with four stretches of 5Gs connected by loops of differing length and capped by 0-4T (labeled LM). Biophysical characterization of 34 LM variants indicates that they fold into predominantly antiparallel GQs—only five variants display significant hybrid/parallel character. PAGE revealed that LM variants are monomolecular and relatively homogeneous. All LM variants display high stability. Increase in the number of 5’-T or loop length diminishes the antiparallel fold and decreases stability of the GQs, with loop length having stronger effect. 3’-T have less effect on the fold and stability. We designed mutants where each 5G stretch was trimmed to 4G – these mutants displayed significantly lower thermal stability (by > 6 °C) suggesting that LM variants indeed contain 5-tetrads. We solved a crystal structure of one of the variants which displayed an interlaced dimer of 5-tetrad hybrid GQs. One G from each monomer participates in the formation of the first G-tetrad in the symmetry generated partner. The 5-tetrad GQs can be great therapeutic targets, as their rarity would lead to a greater selectivity of drugs that target them.

Cancer stands as a pervasive global health challenge and chemotherapy stands as the prevailing approach to treat it. While traditional chemotherapeutic agents have proven effective in cancer treatment, the consequential impact on the physical and psychological well-being of patients remains notably severe. Furthermore, the adaptability of tumor cells to develop resistance against a wide array of chemotherapeutic drugs poses a significant obstacle, ultimately resulting in treatment failures.In this context, chemo-sensitization has gained attention as a strategy using small molecules to enhance cancer cells’ sensitivity to conventional drugs. This approach aims to tackle chemoresistance mechanisms, reduce chemotherapy-induced side effects, and improve clinical outcomes.This presentation explores the growing relevance of G-quadruplex (G4) structures as promising anti-cancer targets. The goal is to boost the antitumor efficacy of standard chemotherapeutics by leveraging G4-interacting molecules, enabling synergistic effects with conventional drugs while minimizing toxicity in healthy cells and overcome drug resistance.