Guanine quadruplexes mediate mitochondrial RNA polymerase pausing

BMC Biology · 2025-05-13 · 4 citations

BACKGROUND: The information content within nucleic acids extends beyond the primary sequence to include secondary structures with functional roles in transcription regulation. Guanine-rich sequences form structures called guanine quadruplexes that result from non-canonical base pairing between guanine residues. These stable guanine quadruplex structures are prevalent in gene promoters in nuclear DNA and are known to be associated with promoter proximal pausing of some genes. However, the transcriptional impact of guanine quadruplexes that form in nascent RNA is poorly understood. RESULTS: We examined mitochondrial RNA polymerase (POLRMT) pausing patterns in primary human skin fibroblast cells using the precision nuclear run-on assay and uncovered over 400 pause sites on the mitochondrial genome. We identified that these pauses frequently occur following guanine-rich sequences where quadruplexes form. Using an in vitro primer extension assay, we show that quadruplexes formed in nascent RNA act as mediators of POLRMT pausing, and in cell-based assays their stabilization disrupts POLRMT transcription. Cells exposed to a guanine-quadruplex stabilizing agent (RHPS4) had diminished mitochondrial gene expression and significantly lowered cellular respiration within 24 h. The resulting ATP stress was sufficient to reduce active transport in renal epithelia. CONCLUSIONS: Our findings connect RNA guanine quadruplex-mediated pausing with the regulation of POLRMT transcription and mitochondrial function. We demonstrate that tuning of quadruplex dynamics in nascent RNA, rather than template DNA upstream of the polymerase, is sufficient to regulate mitochondrial gene expression.

Unlocking the regulatory code of RNA: launching the Human RNome Project

Genome biology · 2025-10-24 · 4 citations

The human RNome, the complete set of RNA molecules in human cells, arises through complex processing and includes diverse molecular species. While research traditionally focuses on four canonical nucleotide residues, the RNome, encompassing over 180 distinct modifications across organisms, with at least 50 in humans, is increasingly recognized. These modifications play critical roles in regulating RNA structure, stability, and function, yet the rules linking their precise locations to biological outcomes remain poorly defined. The Human RNome Project aims to map all RNA modifications, build essential resources, and harness new technologies to transform RNA biology, therapeutic development, agriculture, and even data storage.

Toward standardized epitranscriptome analytics: an inter-laboratory comparison of mass spectrometric detection and quantification of modified ribonucleosides in human RNA

Nucleic Acids Research · 2025-09-05 · 8 citations

The human RNome comprises all forms of RNA and the 50 + chemical structures-the epitranscriptome-that modify them. Understanding the diverse functions of RNA modifications in regulating gene expression and cell phenotype requires technologies such as RNA sequencing-based modification mapping and mass spectrometry-based quantification of modified ribonucleosides. Liquid chromatography-coupled tandem quadrupole mass spectrometry (LC-MS/MS) is the gold standard for detecting and quantifying modified ribonucleosides with accuracy and precision. However, variations in RNA isolation, processing, and LC-MS/MS analysis have hindered reproducibility across laboratories, which is essential for accurate quantification of RNA modifications. As guidance toward harmonization, we report a multi-laboratory comparison of workflows for LC-MS/MS RNA modification analysis. We compared protocols for sample shipment, RNA hydrolysis, LC-MS/MS analysis, and data processing among three laboratories working with the same total RNA samples. We detected and quantified 17 modifications consistently across protocols and operators, with another 7 that were sensitive to experimental conditions, reagent contamination, and ribonucleoside instability, leading to poor precision among laboratories. Agreement among the three labs was strong, with coefficients of variation of 20% and 10% for relative and absolute quantification, respectively. These findings establish a robust and readily adoptable epitranscriptome analytical platform that enables reliable comparisons across laboratories.

Allele-specific silencing of a dominant SETX mutation in familial amyotrophic lateral sclerosis type 4

Human Genetics and Genomics Advances · 2025-04-08 · 1 citations

Amyotrophic lateral sclerosis 4 (ALS4) is an autosomal dominant motor neuron disease that is molecularly characterized by reduced R-loop levels and caused by pathogenic variants in senataxin (SETX). SETX encodes an RNA/DNA helicase that resolves three-stranded nucleic acid structures called R-loops. Currently, there are no disease-modifying therapies available for ALS4. Given that SETX is haplosufficient, removing the product of the mutated allele presents a potential therapeutic strategy. We designed a series of siRNAs to selectively target the RNA transcript from the ALS4 allele containing the c.1166T>C mutation (p.Leu389Ser). Transfection of HEK293 cells with siRNA and plasmids encoding either wild-type or mutant (Leu389Ser) epitope-tagged SETX revealed that three siRNAs specifically reduced mutant SETX protein levels while having minimal effect on the wild-type SETX protein. In ALS4 primary fibroblasts, siRNA treatment silenced the endogenous mutant SETX allele while sparing the wild-type allele and restored R-loop levels in patient cells. Our findings demonstrate that mutant SETX, differing from wild-type by a single nucleotide, can be effectively and specifically silenced by RNA interference.

Allele-specific silencing of a dominant <i>SETX</i> mutation in familial amyotrophic lateral sclerosis type 4

bioRxiv (Cold Spring Harbor Laboratory) · 2024-10-12

ABSTRACT Amyotrophic lateral sclerosis 4 (ALS4) is an autosomal dominant motor neuron disease that is molecularly characterized by reduced R-loop levels and caused by pathogenic variants in senataxin ( SETX ). SETX encodes an RNA/DNA helicase that resolves three-stranded nucleic acid structures called R-loops. Currently, there are no disease-modifying therapies available for ALS4. Given that SETX is haplosufficient, removing the product of the mutated allele presents a potential therapeutic strategy. We designed a series of siRNAs to selectively target the RNA transcript from the ALS4 allele containing the c.1166T&gt;C mutation (p.Leu389Ser). Transfection of HEK293 cells with siRNA and plasmids encoding either wild-type or mutant (Leu389Ser) epitope tagged SETX revealed that three siRNAs specifically reduced mutant SETX protein levels without affecting the wild-type SETX protein. In ALS4 primary fibroblasts, siRNA treatment silenced the endogenous mutant SETX allele, while sparing the wild-type allele, and restored R-loop levels in patient cells. Our findings demonstrate that mutant SETX , differing from wild-type by a single nucleotide, can be effectively and specifically silenced by RNA interference, highlighting the potential of allele-specific siRNA as a therapeutic approach for ALS4.

Isoform Switching Regulates the Response to Ionizing Radiation Through SRSF1

International Journal of Radiation Oncology*Biology*Physics · 2024-03-05 · 3 citations

PURPOSE: This study investigated how isoform switching affects the cellular response to ionizing radiation (IR), an understudied area despite its relevance to radiation therapy in cancer treatment. We aimed to identify changes in transcript isoform expression post-IR exposure and the proteins mediating these changes, with a focus on their potential to modulate radiosensitivity. METHODS AND MATERIALS: Using RNA sequencing, we analyzed the B-cell lines derived from 10 healthy individuals at 3 timepoints, applying the mixture of isoforms algorithm to quantify alternative splicing. We examined RNA binding protein motifs within the sequences of IR-responsive isoforms and validated the serine/arginine-rich splicing factor 1 (SRSF1) as a predominant mediator through RNA immunoprecipitation. We further investigated the effects of SRSF1 on radiosensitivity by RNA interference and by analyzing publicly available data on patients with cancer. RESULTS: We identified ∼1900 radiation-responsive alternatively spliced isoforms. Many isoforms were differentially expressed without changes in their overall gene expression. Over a third of these transcripts underwent exon skipping, while others used proximal last exons. These IR-responsive isoforms tended to be shorter transcripts missing vital domains for preventing apoptosis and promoting cell division but retaining those necessary for DNA repair. Our combined computational, genetic, and molecular analyses identified the proto-oncogene SRSF1 as a mediator of these radiation-induced isoform-switching events that promote apoptosis. After exposure to DNA double-strand break-inducing agents, SRSF1 expression decreased. A reduction in SRSF1 increased radiosensitivity in vitro and among patients with cancer. CONCLUSIONS: We establish a pivotal role for isoform switching in the cellular response to IR and propose SRSF1 as a promising biomarker for assessing radiation therapy effectiveness.

The enhancer RNA, AANCR, regulates APOE expression in astrocytes and microglia

Nucleic Acids Research · 2024-08-20 · 8 citations

Enhancers, critical regulatory elements within the human genome, are often transcribed into enhancer RNAs. The dysregulation of enhancers leads to diseases collectively termed enhanceropathies. While it is known that enhancers play a role in diseases by regulating gene expression, the specific mechanisms by which individual enhancers cause diseases are not well understood. Studies of individual enhancers are needed to fill this gap. This study delves into the role of APOE-activating noncoding RNA, AANCR, in the central nervous system, elucidating its function as a genetic modifier in Alzheimer's Disease. We employed RNA interference, RNaseH-mediated degradation, and single-molecule RNA fluorescence in situ hybridization to demonstrate that mere transcription of AANCR is insufficient; rather, its transcripts are crucial for promoting APOE expression. Our findings revealed that AANCR is induced by ATM-mediated ERK phosphorylation and subsequent AP-1 transcription factor activation. Once activated, AANCR enhances APOE expression, which in turn imparts an inflammatory phenotype to astrocytes. These findings demonstrate that AANCR is a key enhancer RNA in some cell types within the nervous system, pivotal for regulating APOE expression and influencing inflammatory responses, underscoring its potential as a therapeutic target in neurodegenerative diseases.

Supplementary Figures 1-2 from Identification of Novel p53 Target Genes in Ionizing Radiation Response

2023-03-30

&lt;p&gt;Also includes supplementary methods and materials&lt;/p&gt;

Nanopore-based direct sequencing of RNA transcripts with 10 different modified nucleotides reveals gaps in existing technology

G3 Genes Genomes Genetics · 2023-09-01 · 12 citations

RNA undergoes complex posttranscriptional processing including chemical modifications of the nucleotides. The resultant-modified nucleotides are an integral part of RNA sequences that must be considered in studying the biology of RNA and in the design of RNA therapeutics. However, the current "RNA-sequencing" methods primarily sequence complementary DNA rather than RNA itself, which means that the modifications present in RNA are not captured in the sequencing results. Emerging direct RNA-sequencing technologies, such as those offered by Oxford Nanopore, aim to address this limitation. In this study, we synthesized and used Nanopore technology to sequence RNA transcripts consisting of canonical nucleotides and 10 different modifications in various concentrations. The results show that direct RNA sequencing still has a baseline error rate of >10%, and although some modifications can be detected, many remain unidentified. Thus, there is a need to develop sequencing technologies and analysis methods that can comprehensively capture the total complexity of RNA. The RNA sequences obtained through this project are made available for benchmarking analysis methods.

Data from Identification of Novel p53 Target Genes in Ionizing Radiation Response

2023-03-30

&lt;div&gt;Abstract&lt;p&gt;The tumor suppressor p53 plays an essential role in cellular adaptation to stress. In response to ionizing radiation, p53 regulates the transcription of genes in a diverse set of pathways including DNA repair, cell cycle arrest, and apoptosis. Previously, we identified by microarray analysis a set of genes that are transcriptionally activated or repressed in response to radiation exposure. In this study, we use computational methods and molecular techniques, including location analysis (ChIP-on-chip assay), to identify ionizing radiation–responsive genes that are directly regulated by p53. Among the 489 ionizing radiation–responsive genes examined, 38 genes were found to be p53 targets. Some of these genes are previously known to be directly regulated by p53 whereas others are novel p53 targets. We further showed that the novel p53 target genes are transcriptionally regulated by p53. The binding of p53 to promoters of target genes correlated with increased transcript levels of these genes in cells with functional p53. However, p53 binding and subsequent transcriptional activation of these target genes were significantly diminished in cells with mutant p53 and in cells from patients with ataxia telangiectasia, which have impaired p53 activation following ionizing radiation exposure. Identification and characterization of ionizing radiation–responsive p53 target genes extend our knowledge of the diverse role that p53 plays in the DNA damage response.&lt;/p&gt;&lt;/div&gt;