TERRA and RAD51AP1 promote alternative lengthening of telomeres through an R- to D-loop switch.

Alternative lengthening of telomeres (ALT), a telomerase-independent process maintaining telomeres, is mediated by break-induced replication (BIR). RAD52 promotes ALT by facilitating D-loop formation, but ALT also occurs through a RAD52-independent BIR pathway. Here, we show that the telomere non-coding RNA TERRA forms dynamic telomeric R-loops and contributes to ALT activity in RAD52 knockout cells. TERRA forms R-loops in vitro and at telomeres in a RAD51AP1-dependent manner. The formation of R-loops by TERRA increases G-quadruplexes (G4s) at telomeres. G4 stabilization enhances ALT even when TERRA is depleted, suggesting that G4s act downstream of R-loops to promote BIR. In vitro, the telomeric R-loops assembled by TERRA and RAD51AP1 generate G4s, which persist after R-loop resolution and allow formation of telomeric D-loops without RAD52. Thus, the dynamic telomeric R-loops formed by TERRA and RAD51AP1 enable the RAD52-independent ALT pathway, and G4s orchestrate an R- to D-loop switch at telomeres to stimulate BIR.

Replication protein A associates with nucleolar R loops and regulates rRNA transcription and nucleolar morphology.

The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated "promoter and pre-rRNA antisense" RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.

R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.

A non-coding RNASEH1 gene variant associates with type 1 diabetes and interacts with HLA tagSNPs in families from Colombia.

OBJECTIVES: RNASEH1 gene has recently been associated with type 1 diabetes (T1D) in Colombia. The purpose of this study was to fine mapping the putative functional variant in RNASEH1 and testing its interaction with HLA tagSNPs. METHODS: Two-hundred nuclear families with T1D were included in this study. Probands were tested for GAD65 and IA-2 autoantibodies. Genotyping was performed using 20 coding tagSNPs uncovered through Sanger sequencing (N = 96), in addition to 23 tagSNPs chosen from 1000genomes to cover the extent of the gene region. Also, 45 tagSNPs for classic HLA alleles associated with T1D were also genotyped. The transmission disequilibrium test (TDT) was used to test for association and a multiple testing correction was made using permutation. Interaction between RNASEH1 variants and HLA was evaluated by means of the M-TDT test. RESULTS: We identified 20 variants (15 were novel) in the 96 patients sequenced. None of these variants were in linkage disequilibrium. In total, 43 RNASEH1 variants were genotyped in the 200 families. Association between T1D and rs7607888 was identified (P = .002). Haplotype analysis involving rs7607888 variant revealed even stronger association with T1D (most significative P = .0003). HLA tagSNPs displayed stronger associations (OR = 6.39, 95% CI = 4.33-9.44, P-value = 9.74E-28). Finally, we found several statistically significant interactions of HLA variants with rs7607888 (P-value ranged from 8.77E-04 to 5.33E-12). CONCLUSION: Our results verify the association of rs7607888 in RNASEH1 gene with T1D. It is also shown in the interaction between RNASEH1 and HLA for conveying risk to T1D in Northwest Colombia. Work is underway aiming to identify the actual classic HLA alleles associated with the tagSNPs tested here.

RNASEH1 gene variants are associated with autoimmune type 1 diabetes in Colombia.

BACKGROUND: In a previous work, we found linkage and association of type 1 diabetes (T1D) to a 12 known gene region at chromosome 2p25 in Colombian families. Here, we present further work on this candidate region. MATERIALS AND METHODS: Seventeen SNPs located on the 12 candidate genes, in 100 familial trios set, were tested by ARMS-tetraprimer-PCR or PCR-RFLP. Five extra SNPs in the vicinity of rs10186193 were typed. A replica phase included 97 novel familial trios, in whom diabetes-related auto-antibodies (AABs) were tested in sera of the patients. In addition to transmission disequilibrium tests, haplotype analyses were carried out using the unphased software. RESULTS: SNP rs10186193 (at RNASEH1 gene) showed association with T1D (P = 0.005). The additional five SNPs revealed that rs7607888 (P = 2.03 × 10-7), rs55981318 (P = 0.018), and rs1136545 (P = 1.93 × 10-9) were also associated with T1D. Haplotype analysis showed association for rs55981318-rs10186193 (P = 0.0005), rs7563960-rs7607888 (P = 0.0007), rs7607888-rs1136545 (P = 9.21 × 10-10), and rs1136545-rs11538545 (P = 6.67 × 10-8). In contrast, the new set of 97 familial trios tested for SNPs rs55981318, rs10186193, and rs7607888 did not support the previous finding; however, by combining the sample (197 trios), evidence of association of T1D with rs55981318 and rs7607888 was conclusive. In addition, a two-loci haplotype analysis of the combined sample showed significant association of RNASEH1 with T1D (P = 3.1 × 10-5). CONCLUSION: In conclusion, our analyses suggest that RNASEH1 gene variants associate with susceptibility/protection to T1D in Colombia.

Comprehensive analysis identifies long non-coding RNA RNASEH1-AS1 as a potential prognostic biomarker and oncogenic target in hepatocellular carcinoma.

RNASEH1-AS1, a long non-coding RNA (lncRNA) divergently transcribed from the antisense strand of its neighboring protein-coding gene ribonuclease H1 (RNASEH1), has recently been demonstrated to be involved in tumor progression. However, the association between RNASEH1-AS1 and hepatocellular carcinoma (HCC) remains unclear. In the present study, first, the expression of RNASEH1-AS1 in HCC and its correlation with clinicopathological features, prognosis, diagnosis, immune cell infiltration of HCC patients was inspected using relevant R packages based on The Cancer Genome Atlas (TCGA) data. RNASEH1-AS1 was found to be up-regulated in most cancer types, including HCC, and its overexpression was significantly associated with histologic grade and AFP level as well as poor prognosis, and was an independent risk factor affecting overall survival with good diagnostic and prognostic values for HCC. RNASEH1-AS1 was inversely associated with the infiltration of most immune cell types, including plasmacytoid dendritic cells (pDC), B cells and neutrophils. Second, a total of 1109 positively co-expressed genes (PCEGs) of RNASEH1-AS1 were screened out in HCC by correlation analysis in batches (|Spearman's r| >0.4 and adjusted P value <0.01). GO and KEGG enrichment analysis indicated that PCEGs of RNASEH1-AS1 were mainly related to RNA processing, ribosome biogenesis, transcription and histone acetylation. The top 10 hub genes (EIF4A3, WDR43, WDR12, DKC1, NAT10, UTP18, DDX18, BYSL, DDX10, PDCD11) were identified by constructing the protein-protein interaction (PPI) network, and they were all highly expressed in HCC and positively correlated with histological grade. Third, a risk model was constructed based on four RNASEH1-AS1-related hub genes (EIF4A3, WDR12, DKC1, and NAT10) with good prognostic predictive potential via univariate Cox and the least absolute selection operator (LASSO) regression analysis. Fourth, experimental validation revealed that RNASEH1-AS1 was significantly elevated in HCC tissues and several cell lines, and its knockdown could suppress the proliferation, migration, and invasion of HCC cells. Finally, mechanistic studies demonstrated that the stability of RNASEH1-AS1 could be regulated by DKC1 via their direct interaction. Taken together, RNASEH1-AS1 may serve as a potential prognostic and diagnostic biomarker and oncogenic lncRNA for HCC.

A pan-cancer analysis of RNASEH1, a potential regulator of the tumor microenvironment.

BACKGROUND: RNASEH1 (Ribonuclease H1) encodes an endonuclease that specifically degrades the RNA of RNA-DNA hybrids and acts in DNA replication and repair. Although there are many studies on RNASEH1, the research of RNASEH1 in cancers is still insufficient. Therefore, in order to clarify the physiological mechanism of RNASEH1 in tumor cells, we evaluated the role of RNASEH1 by combining The Cancer Genome Atlas (TCGA) pan-cancer data and Genotype-Tissue Expression (GTEx) normal tissue data. METHODS: RNASEH1 expression was analyzed by using RNAseq data from TCGA and the GTEx database. The Human Protein Atlas (HPA), GeneCards and STRING database were used to explore the protein information of RNASEH1. The prognostic value of RNASEH1 was analyzed by using the clinical survival data from TCGA. Differential analysis of RNASEH1 in different cancers was performed by using R package "DESeq2", and enrichment analysis of RNASEH1 was conducted by using R package "clusterProfiler". We downloaded the immune cell infiltration score of TCGA samples from published articles and online databases, and the correlation analysis between immune cell infiltration levels and RNASEH1 expression was performed. Not only that, we further evaluated the association of RNASEH1 with immune activating genes, immunosuppressive genes, chemokines and chemokine receptors. At the end of the article, the differential expression of RNASEH1 in pan-cancer was validated by using GSE54129, GSE40595, GSE90627, GSE106937, GSE145976 and GSE18672, and qRT-PCR was also performed for verification. FINDINGS: RNASEH1 was significantly overexpressed in 19 cancers and the overexpression was closely correlated with poor prognosis. Moreover, the expression of RNASEH1 was significantly correlated with the regulation of the tumor microenvironment. In addition, RNASEH1 expression was closely associated with immune cell infiltration, immune checkpoints, immune activators, immunosuppressive factors, chemokines and chemokine receptors. Finally, RNASEH1 also was closely associated with DNA-related physiological activities and mitochondrial-related physiological activities. INTERPRETATION: Our studying suggests that RNASEH1 is a potential cancer biomarker. And RNASEH1 may be able to regulate the tumor microenvironment by regulating the relevant physiological activities of mitochondrial and thereby regulating the occurrence and development of tumors. Thus, it could be used to develop new-targeted drugs of tumor therapy.

Mapping R-Loops Using Catalytically Inactive RNaseH1 (R-ChIP).

R-loops, three-stranded structures containing double-stranded DNA invaded by single-stranded RNA, have been linked to diverse biological processes. They play important roles in regulating gene regulation and DNA repair, contributing to a wide range of diseases. Understanding the formation and dynamic regulation of R-loops is thus a gateway to address many fundamental questions in regulatory biology, which requires the elucidation of the R-loop landscape at the genome scale. To aid in such efforts, this article provides an overview on R-loop mapping strategies along with a detailed protocol based on the use of catalytically inactive RNaseH1, an evolutionarily conserved protein responsible for R-loop recognition and resolution.

Case Report: Rare Homozygous RNASEH1 Mutations Associated With Adult-Onset Mitochondrial Encephalomyopathy and Multiple Mitochondrial DNA Deletions.

Mitochondrial DNA (mtDNA) maintenance disorders embrace a broad range of clinical syndromes distinguished by the evidence of mtDNA depletion and/or deletions in affected tissues. Among the nuclear genes associated with mtDNA maintenance disorders, RNASEH1 mutations produce a homogeneous phenotype, with progressive external ophthalmoplegia (PEO), ptosis, limb weakness, cerebellar ataxia, and dysphagia. The encoded enzyme, ribonuclease H1, is involved in mtDNA replication, whose impairment leads to an increase in replication intermediates resulting from mtDNA replication slowdown. Here, we describe two unrelated Italian probands (Patient 1 and Patient 2) affected by chronic PEO, ptosis, and muscle weakness. Cerebellar features and severe dysphagia requiring enteral feeding were observed in one patient. In both cases, muscle biopsy revealed diffuse mitochondrial abnormalities and multiple mtDNA deletions. A targeted next-generation sequencing analysis revealed the homozygous RNASEH1 mutations c.129-3C>G and c.424G>A in patients 1 and 2, respectively. The c.129-3C>G substitution has never been described as disease-related and resulted in the loss of exon 2 in Patient 1 muscle RNASEH1 transcript. Overall, we recommend implementing the use of high-throughput sequencing approaches in the clinical setting to reach genetic diagnosis in case of suspected presentations with impaired mtDNA homeostasis.

Long noncoding RNASEH1-AS1 exacerbates the progression of non-small cell lung cancer by acting as a ceRNA to regulate microRNA-516a-5p/FOXK1 and thereby activating the Wnt/ß-catenin signaling pathway.

BACKGROUND: Till now, no study has focused on the functions of RNASEH1 antisense RNA 1 (RNASEH1-AS1) in non-small cell lung cancer (NSCLC). Accordingly, we measured the expression of RNASEH1-AS1 in NSCLC and characterized its functions in detail. Finally, our research elucidated the mechanisms that occurred downstream of RNASEH1-AS1. METHODS: RNASEH1-AS1 expression was examined utilizing TCGA database and qRT-PCR. Functional experiments were conducted to study the tumor-associated functions of RNASEH1-AS1. The targeting relationship among RNASEH1-AS1, microRNA-516a-5p (miR-516a-5p), and forkhead box K1 (FOXK1) was revealed utilizing RNA immunoprecipitation and luciferase reporter assays. RESULTS: Utilizing TCGA database and our own cohort, we found a significantly increased level of RNASEH1-AS1 in NSCLC. The high level of RNASEH1-AS1 was markedly related with poor clinical outcomes. Knockdown of RNASEH1-AS1 expression inhibited NSCLC cell growth, metastatic capacities, and epithelial-mesenchymal transition and promoted the apoptosis in vitro, whereas RNASEH1-AS1 overexpression exerted the opposite effects. Additionally, knocking down RNASEH1-AS1 expression suppressed tumor growth in vivo. RNASEH1-AS1 was confirmed to act as a miR-516a-5p sponge, consequently upregulating FOXK1 expression in NSCLC cells. As revealed by the subsequent rescue experiments, the miR-516a-5p/FOXK1 axis served as a downstream effector of RNASEH1-AS1. In addition, by controlling the miR-516a-5p/FOXK1 axis, RNASEH1-AS1 was capable of activating the Wnt/ß-catenin pathway. CONCLUSION: RNASEH1-AS1 exacerbated the oncogenicity of NSCLC by affecting the miR-516a-5p/FOXK1 axis and consequently promoting the activation of Wnt/ß-catenin pathway. Our newly identified RNASEH1-AS1/miR-516a-5p/FOXK1/Wnt/ß-catenin network may offer an interesting foundation for NSCLC treatment in the clinic.

BRCA2 deficiency reveals that oxidative stress impairs RNaseH1 function to cripple mitochondrial DNA maintenance.

Oxidative stress is a ubiquitous cellular challenge implicated in aging, neurodegeneration, and cancer. By studying pathogenic mutations in the tumor suppressor BRCA2, we identify a general mechanism by which oxidative stress restricts mitochondrial (mt)DNA replication. BRCA2 inactivation induces R-loop accumulation in the mtDNA regulatory region and diminishes mtDNA replication initiation. In BRCA2-deficient cells, intracellular reactive oxygen species (ROS) are elevated, and ROS scavengers suppress the mtDNA defects. Conversely, wild-type cells exposed to oxidative stress by pharmacologic or genetic manipulation phenocopy these defects. Mechanistically, we find that 8-oxoguanine accumulation in mtDNA caused by oxidative stress suffices to impair recruitment of the mitochondrial enzyme RNaseH1 to sites of R-loop accrual, restricting mtDNA replication initiation. Thus, oxidative stress impairs RNaseH1 function to cripple mtDNA maintenance. Our findings highlight a molecular mechanism that links oxidative stress to mitochondrial dysfunction and is elicited by the inactivation of genes implicated in neurodegeneration and cancer.

Progressive External Ophthalmoplegia in Polish Patients-From Clinical Evaluation to Genetic Confirmation.

Mitochondrial encephalomyopathies comprise a group of heterogeneous disorders resulting from impaired oxidative phosphorylation (OxPhos). Among a variety of symptoms progressive external ophthalmoplegia (PEO) seems to be the most common. The aim of this study is to present clinical and genetic characteristics of Polish patients with PEO. Clinical, electrophysiological, neuroradiological, and morphological data of 84 patients were analyzed. Genetic studies of mitochondrial DNA (mtDNA) were performed in all patients. Among nuclear DNA (nDNA) genes POLG was sequenced in 41 patients, TWNK (C10orf2) in 13 patients, and RNASEH1 in 2 patients. Total of 27 patients were included in the chronic progressive external ophthalmoplegia (CPEO) group, 24 in the CPEO+ group. Twenty-six patients had mitochondrial encephalomyopathy (ME), six patients Kearns-Sayre syndrome (KSS), and one patient sensory ataxic neuropathy, dysarthria, ophthalmoparesis (SANDO) syndrome. Genetic analysis of nDNA genes revealed the presence of pathogenic or possibly pathogenic variants in the POLG gene in nine patients, the TWNK gene in five patients and the RNASEH1 gene in two patients. Detailed patients' history and careful assessment of family history are essential in the diagnostic work-up. Genetic studies of both mtDNA and nDNA are necessary for the final diagnosis of progressive external ophthalmoplegia and for genetic counseling.

Identification and Characterization of New RNASEH1 Mutations Associated With PEO Syndrome and Multiple Mitochondrial DNA Deletions.

Mitochondrial DNA (mtDNA) depletion and deletion syndrome encompasses a group of disorders caused by mutations in genes involved in mtDNA replication and maintenance. The clinical phenotype ranges from fatal infantile hepatocerebral forms to mild adult onset progressive external ophthalmoplegia (PEO). We report the case of a patient with PEO and multiple mtDNA deletions, with two new homozygous mutations in RNASEH1. The first mutation (c.487T>C) is located in the same catalytic domain as the four previously reported mutations, and the second (c.258_260del) is located in the connection domain, where no mutations have been reported. In silico study of the mutations predicted only the first mutation as pathogenic, but functional studies showed that both mutations cause loss of ribonuclease H1 activity. mtDNA replication dysfunction was demonstrated in patient fibroblasts, which were unable to recover normal mtDNA copy number after ethidium bromide-induced mtDNA depletion. Our results demonstrate the pathogenicity of two new RNASEH1 variants found in a patient with PEO syndrome, multiple deletions, and mild mitochondrial myopathy.

Posible implicación del gen RNASEH1 en la etiología de Diabetes Mellitus tipo 1 / Possible implication of the RNASEH1 gene in the etiology of type 1 diabetes mellitus

Las enfermedades complejas se caracterizan porque presentan varios genes además de factores ambientales implicados en su etiología. Las bases genéticas de la diabetes mellitus tipo 1 (T1D) supone un efecto mayor del complejo HLA que interactúa con otros genes y con el ambiente. Mucho se ha descrito acerca de la posible participación de las infecciones virales como desencadenadores de T1D. En esta revisión exploramos los posibles mecanismos por los cuales el gen RNASEH1 podría estar participando en la etiología de T1D, a partir de una infección viral. El gen RNASEH1 se localiza en la región cromosómica 2p25, la cual ha sido recientemente implicada por nosotros en la susceptibilidad a T1D. Este gen ha sido implicado en la enfermedad mediante análisis genético. Acá pretendemos dar sentido biológico a los datos genéticos. Considerando que la enfermedad es multifactorial, este planteamiento no excluye la participación de otros genes u otros factores ambientales.

Complex disorders are characterized by presenting many genes and other environmental factors implicated in their etiology. The genetic bases of type 1 diabetes mellitus (T1D) suppose a major effect of the HLA complex which interacts with other genes and the environment. Much has been written about the possible implication of viral infections as triggers of T1D. This review explores the mechanisms by which the RNASEH1 gene could be involved in the etiology of T1D, due to a viral infection. The RNASEH1 gene is located in chromosome 2p25, which has been recently implicated in the susceptibility to T1D by the authors, through genetic analysis.This text hopes to establish a biological context for the genetic data. Taking into account that this is a multifactorial disease, this approach does not exclude the eventual participation of other genes or environmental factors.