rG4: A Key Factor in RNA G-Quadruplex Biology and Health

RNA G-quadruplexes (rG4s) are specialized secondary structures in RNA molecules that play roles in gene regulation, translation, and disease. Forming in guanine-rich sequences, they influence cellular processes by altering RNA stability, localization, and interactions with proteins. Their presence across various organisms suggests a fundamental biological function.

Understanding rG4s is crucial as research links them to both normal cellular function and diseases such as cancer and neurodegenerative disorders. Scientists are investigating their impact on gene expression and disease mechanisms.

Molecular Architecture Of G-Quadruplexes In RNA

RNA G-quadruplexes (rG4s) are non-canonical secondary structures arising from guanine-rich sequences folding into stacked tetrads stabilized by Hoogsteen hydrogen bonding. Unlike Watson-Crick base pairing in double-stranded RNA, these structures rely on guanine residues forming planar G-quartets, further stabilized by monovalent cations like potassium (K⁺) and sodium (Na⁺). These cations fit within the central channel of the quadruplex, reinforcing stacking interactions and structural integrity.

The structural diversity of rG4s depends on sequence composition and loop length. These loops, connecting guanine tetrads, influence topology and stability, resulting in parallel, antiparallel, or hybrid conformations. In RNA, the parallel topology is most common due to the rigidity of the ribose sugar. This differs from DNA G-quadruplexes, where antiparallel and hybrid forms are more frequent. Loop regions also affect interactions with proteins and small molecules, shaping rG4 function.

The thermodynamic stability of rG4s is a key factor in their biological role. Spectroscopic techniques such as circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy show that RNA G-quadruplexes are more thermally stable than their DNA counterparts. This stability is attributed to the 2′-hydroxyl group of the ribose sugar, which enhances hydrogen bonding and stacking interactions. Stability can be further modulated by RNA-binding proteins, post-transcriptional modifications like methylation, and the local cellular environment, including ionic concentrations and molecular crowding.

Laboratory Approaches For Detection

Detecting rG4s requires specialized techniques that capture their structural and functional properties in different biological contexts. Researchers use sequencing-based, biophysical, and chemical probing methods to identify and characterize these structures with high specificity.

rG4-Seq

rG4-Seq is a high-throughput sequencing method that maps rG4 structures across the transcriptome. It employs G-quadruplex-stabilizing ligands such as pyridostatin (PDS) or Phen-DC3, which bind to rG4s and induce structural stabilization. During library preparation, reverse transcriptase stalls at these stabilized rG4s, creating characteristic stops in cDNA synthesis. These interruptions are detected through next-generation sequencing, pinpointing rG4-forming regions with nucleotide-level resolution.

This method provides transcriptome-wide coverage, identifying rG4s in various RNA species, including messenger RNA (mRNA), long non-coding RNA (lncRNA), and microRNA (miRNA). Studies using rG4-Seq reveal that rG4s are enriched in untranslated regions (UTRs) and coding sequences, suggesting roles in post-transcriptional regulation. However, potential biases from stabilizing ligands and difficulty distinguishing transient from highly stable rG4s remain challenges. Despite these limitations, rG4-Seq is a powerful tool for mapping rG4 landscapes in different cellular conditions.

In Vitro Biophysical Tools

Biophysical techniques provide direct evidence of rG4 formation and stability. Circular dichroism (CD) spectroscopy determines rG4 topology based on spectral signatures. Parallel rG4s exhibit a positive peak at 260 nm and a negative peak at 240 nm, while antiparallel forms show a positive peak at 295 nm. This method assesses folding preferences under different ionic conditions and in the presence of stabilizing ligands.

Nuclear magnetic resonance (NMR) spectroscopy offers atomic-level resolution, analyzing imino proton resonances to confirm Hoogsteen hydrogen bonding. Additional techniques like fluorescence resonance energy transfer (FRET) and isothermal titration calorimetry (ITC) study rG4 stability and ligand binding affinities. These biophysical tools validate rG4 formation and screen potential rG4-targeting compounds for drug discovery.

Selective Chemical Probing

Chemical probing methods use small molecules or modified nucleotides to selectively detect rG4 structures in living cells. N-methyl mesoporphyrin IX (NMM) and Thioflavin T (ThT) fluoresce upon binding to rG4s, enabling real-time monitoring in live-cell imaging studies.

Dimethyl sulfate (DMS) probing exploits the differential reactivity of guanine residues. In single-stranded RNA, guanines are methylated by DMS, whereas those involved in G-quartets are protected. Comparing DMS reactivity before and after rG4 stabilization reveals structural features. SHALiPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension) maps rG4s by detecting conformational constraints imposed by the quadruplex structure.

These chemical approaches complement sequencing and biophysical methods by providing functional insights into rG4 behavior in physiological conditions.

Impact On Gene Regulation And Protein Synthesis

RNA G-quadruplexes (rG4s) influence gene regulation and protein synthesis by altering RNA stability, translation efficiency, and ribosome dynamics. In untranslated regions (UTRs) of messenger RNA (mRNA), they modulate translation rates by either facilitating or impeding ribosome progression. In 5′ UTRs, rG4s often repress translation by forming stable structures that hinder ribosome scanning and initiation. This effect has been observed in oncogenes such as NRAS, where an rG4 structure reduces protein synthesis by obstructing translation initiation. Conversely, rG4s can enhance translation by recruiting initiation factors or interacting with helicases that resolve the structure.

Within coding sequences, rG4s can slow ribosome elongation, particularly in stress response genes, where controlled translation is beneficial. Specialized helicases such as DHX36 unwind rG4s to facilitate ribosomal progression. The balance between rG4 stability and helicase activity determines whether translation is suppressed or fine-tuned to meet cellular demands.

Beyond translation, rG4s affect mRNA stability and degradation. In the 3′ UTR, they influence interactions with RNA-binding proteins and microRNAs, impacting transcript half-life. rG4s in key metabolic regulators affect decay rates, ensuring tight control of protein levels. The interplay between rG4s and proteins such as AUF1 or hnRNP A1 can either stabilize or destabilize mRNA, depending on the cellular context.

Variation In Different Tissues

The distribution and function of rG4s vary among tissues due to differences in gene expression, RNA-binding proteins, and cellular conditions. In rapidly proliferating tissues such as the intestinal epithelium and bone marrow, rG4s regulate cell cycle and proliferation genes. Their presence suggests a role in fine-tuning gene expression to prevent uncontrolled growth or premature differentiation.

Neural tissues exhibit a distinct rG4 landscape, particularly in transcripts linked to synaptic plasticity and neuronal signaling. The brain’s complex transcriptome and long-lived neuronal mRNAs create an environment where rG4 regulation influences long-term gene expression. rG4s are found in mRNAs encoding neurotransmitter receptors and axon guidance proteins, affecting synaptic function and memory. Specialized helicases like DHX36 modulate rG4 dynamics to maintain neural homeostasis. Disruptions in these mechanisms have been linked to neurodegenerative diseases.

Potential Connections To Human Health

RNA G-quadruplexes (rG4s) are linked to diseases such as cancer, neurodegenerative disorders, and viral infections. Many oncogenes, including MYC, KRAS, and BCL-2, contain rG4-forming sequences that regulate translation and tumor progression. The ability of rG4s to modulate ribosome activity makes them therapeutic targets. Small molecules like TMPyP4 and Phen-DC3 selectively bind rG4s, reducing oncogene expression and showing promise in preclinical cancer models.

Beyond cancer, rG4s are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat expansions in genes like C9orf72 form stable rG4 structures that interfere with RNA metabolism and promote toxic protein aggregates. This misregulation disrupts RNA-binding protein interactions, contributing to neuronal dysfunction.

Viruses also exploit host rG4s to enhance replication. Viral genomes, including HIV and SARS-CoV-2, contain G-rich regions that form quadruplexes, influencing gene expression and immune evasion. Targeting rG4s offers a potential therapeutic strategy for cancer, neurodegenerative diseases, and viral infections.