In Silico Characterization of RNASEH2A Pathogenic Variants and Identification of Novel Splice Site Donor Variant c.549+1G>T in Indian Population.

Background Aicardi-Goutieres syndrome (AGS) is a genetic disorder that has variable manifestations including neurological, immunological, and sometimes other system involvement in various combinations. Considering the high genetic and clinical diversity of AGS and the importance of RNASEH2 complex in the biological system, it is important to take a systematic approach to delineate the genetic diagnosis and impact of missense mutations. Methods Clinical targeted gene sequencing followed by Sanger validation was performed in an individual with the clinical features of AGS. Protein modeling studies of all the reported RNASEH2A missense variants till date were performed using freely available web servers BioGrid, ShinyGO. Protein structures were visualized using Pymol. Results and discussion We identified a novel homozygous splice site donor variant c.549+1G>T in RNASEH2A. Furthermore protein-interactome studies identifiedpotential genetic interactors that include RNASEH2A, RNASEH2B, TYMS, RNASEH2C, RPA1, ORC3, ORC2, CDC6, PCNA, LIG1, PRIM1, RFC2, DUT, GINS1, MCM7, FEN1, MCM4, GINS2, CDK4, and MCM5. Identified genes were mapped to specific pathways using SHINY GO. DNA replication and cell cycle, centrosome cycle, post-replication repair, nucleic acid and metabolic process, cellular response to stress, DNA metabolic process, nucleic acid phosphodiester bond hydrolysis, RNA phosphodiester bond hydrolysis, and DNA biosynthetic process were identified as the linked pathways with the prioritized genes. Conclusion In conclusion, a sophisticated genotype and phenotype correlation followed by linking the genes to the key biological pathways opens new avenues to understand disease pathology and plan for therapeutic interventions.

Hypoxia-induced RNASEH2A limits activation of cGAS-STING signaling in HCC and predicts poor prognosis.

BACKGROUND: Hypoxia is a hallmark of solid cancers, including hepatocellular carcinoma (HCC). There is scarce information about how hypoxia avoids immunologic stress and maintains a cancer-promoting microenvironment. METHODS: The Cancer Genome Atlas, RNA-seq data, and Oncomine database were used to discover the correlation of RNASEH2A with tumor progression; then expression of RNASEH2A mRNA and protein were detected in HCC tissues and cells subjected to hypoxia or with the treatment of CoCl2 via real-time quantitative polymerase chain reaction and immunochemistry assays. Finally, the effect of RNASEH2A on cell proliferation and the involved signaling pathway was explored further. RESULTS: RNASEH2A was positively correlated with tumor grade, size, vascular invasion, and poor prognosis. The expression of RNASEH2A mRNA and protein were increased and dependent on hypoxia-inducible factor 2α in HCC tissues and cell lines. Knockout of RNASEH2A in HCC cells greatly reduced cell proliferation and induced the transcription of multiple cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) targeted type 1 interferon-related genes, including IFIT1, USP18, and CXCL10, which suggests knockout of RNASEH2A may produce immunologic stress and tumor suppressive effects. CONCLUSIONS: RNASEH2A plays a critical role and potentially predicts patient outcomes in HCC, which uncovers a new mechanism that RNASEH2A contributes to limit immunologic stress of cancer cells in the context of hypoxia.

Long noncoding RNA LINC01287 promotes proliferation and inhibits apoptosis of lung adenocarcinoma cells via the miR-3529-5p/RNASEH2A axis under the competitive endogenous RNA pattern.

Lung adenocarcinoma (LUAD) is regarded as the most common type of lung cancer. The molecular targeted therapies for LUAD have being extensively studied. Ribonuclease H2 subunit A (RNASEH2A) is a nucleotide degrading enzyme gene that exerts great influence on cell proliferation, DNA replication and genomic stability. According to bioinformatics analysis, RNASEH2A expression in LUAD tissues is predicted to be upregulated and high expression of RNASEH2A might be related to lower survival rate in LUAD patients. In the present study, we investigated functions of RNASEH2A in LUAD. The mRNA RNASEH2A showed high expression in LUAD cells, and its knockdown inhibited proliferation and induced apoptosis in LUAD cells. RNASEH2A was found to be a target gene of microRNA miR-3529-5p after their expression levels and interaction being examined. Long noncoding RNA LINC01287 upregulated RNASEH2A expression in LUAD cells by combining with miR-3529-5p in a competitive way. Rescue assays revealed that the overexpression of RNASEH2A reversed the suppression of cell proliferation and the promotion of cell apoptosis induced by miR-3529-5p overexpression or LINC01287 knockdown. Finally, forkhead box A1 (FOXA1) interacted with RNASEH2A promoter and LINC01287 promoter to upregulate the expression levels of RNASEH2A and LINC01287 in LUAD cells. Overall, FOXA1-induced LINC01287 serves as a competing endogenous RNA to promote proliferation and inhibit apoptosis of LUAD cells via upregulation of RNASEH2A expression at the posttranscriptional level by competitively combining with miR-3529-5p.

Gene Co-Expression Analysis of Human RNASEH2A Reveals Functional Networks Associated with DNA Replication, DNA Damage Response, and Cell Cycle Regulation.

Ribonuclease (RNase) H2 is a key enzyme for the removal of RNA found in DNA-RNA hybrids, playing a fundamental role in biological processes such as DNA replication, telomere maintenance, and DNA damage repair. RNase H2 is a trimer composed of three subunits, RNASEH2A being the catalytic subunit. RNASEH2A expression levels have been shown to be upregulated in transformed and cancer cells. In this study, we used a bioinformatics approach to identify RNASEH2A co-expressed genes in different human tissues to underscore biological processes associated with RNASEH2A expression. Our analysis shows functional networks for RNASEH2A involvement such as DNA replication and DNA damage response and a novel putative functional network of cell cycle regulation. Further bioinformatics investigation showed increased gene expression in different types of actively cycling cells and tissues, particularly in several cancers, supporting a biological role for RNASEH2A but not for the other two subunits of RNase H2 in cell proliferation. Mass spectrometry analysis of RNASEH2A-bound proteins identified players functioning in cell cycle regulation. Additional bioinformatic analysis showed that RNASEH2A correlates with cancer progression and cell cycle related genes in Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) Pan Cancer datasets and supported our mass spectrometry findings.

Ribonuclease H2 Subunit A impacts invasiveness and chemoresistance resulting in poor survivability of breast cancer in ER dependent manner.

Ribonuclease H2 subunit A (RNASEH2A), a member of the RNase HII family, acts in DNA replication by mediating removal of lagging-strand Okazaki fragment RNA primers. We explored the roles of RNASEH2A in the prognosis of breast cancer, specifically in relation to proliferation, invasiveness, and sensitivity to cytotoxins of cells in the estrogen receptor (ER)-positive MCF-7 and the ER-negative MDA-MB-231 breast cancer cell lines. We collected 26 datasets from around the world, comprising 7815 accessible cases. In these datasets, we probed the association between expression of RNASEH2A and clinical parameters, primarily by inhibiting the expression of RNASEH2A with siRNAs. Such inhibition inhibited the growth and invasiveness of MCF-7 cells. Independent and pooled Kaplan-Meier and Cox analyses revealed that RNASEH2A overexpression was associated with aggressiveness and poor outcomes in a dose-dependent manner in breast cancers of ER-positive subtypes, but not with ER-negative subtypes. The prognostic performance of RNASEH2A mRNA in ER-positive breast cancer was comparable to that for other gene signatures, such as the 21-gene recurrence score. Overexpression of RNASEH2A was also positively associated with cancer cell resistance to chemotherapy; inhibition of RNASEH2A by siRNA enhanced the chemosensitivity in an in vitro study. Bioinformatic analyses indicated that the ER may modulate RNASEH2A action in mitosis, DNA repair, and differentiation through transcriptional regulation. RNASEH2A may be a useful prognostic and predictive biomarker in ER-positive breast cancer and may serve as a therapeutic target for the treatment of ER-positive breast cancer.

Genome-Wide Profiling of Cervical RNA-Binding Proteins Identifies Human Papillomavirus Regulation of RNASEH2A Expression by Viral E7 and E2F1.

RNA-binding proteins (RBPs) control mRNA processing, stability, transport, editing, and translation. We recently conducted transcriptome analyses comparing normal (i.e., healthy) cervical tissue samples with human papillomavirus (HPV)-positive cervical cancer tissue samples and identified 614 differentially expressed protein-coding transcripts which are enriched in cancer-related pathways and consist of 95 known RBPs. We verified the altered expression of 26 genes with a cohort of 72 cervical samples, including 24 normal cervical samples, 25 cervical intraepithelial neoplasia grade 2 (CIN2) and CIN3 samples, and 23 cervical cancer tissue samples. LY6K (lymphocyte antigen 6 complex locus K), FAM83A (family member with sequence similarity 83), CELSR3, ASF1B, IQGAP3, SEMA3F, CLDN10, MSX1, CXCL5, ASRGL1, ELAVL2, GRB7, KHSRP, NOVA1, PTBP1, and RNASEH2A were identified as novel candidate genes associated with cervical lesion progression and carcinogenesis. HPV16 or HPV18 infection was found to alter the expression of 8 RBP genes (CDKN2A, ELAVL2, GRB7, HSPB1, KHSRP, NOVA1, PTBP1, and RNASEH2A) in human vaginal and foreskin keratinocytes. Both viral E6 and E7 decreased NOVA1 expression, but only E7 increased the expression of RNASEH2A in an E2F1-dependent manner. Proliferating cell nuclear antigen (PCNA) directs RNASEH2 activity with respect to DNA replication by removing the RNA primers to promote Okazaki fragment maturation, and two factors are closely associated with neoplasia progression. Therefore, we predict that the induction of expression of RNASEH2A via viral E7 and E2F1 may promote DNA replication and cancer cell proliferation.IMPORTANCE High-risk HPV infections lead to development of cervical cancer. This study identified the differential expression of 16 novel genes (LY6K, FAM83A, CELSR3, ASF1B, IQGAP3, SEMA3F, CLDN10, MSX1, CXCL5, ASRGL1, ELAVL2, GRB7, KHSRP, NOVA1, PTBP1, and RNASEH2A) in HPV-infected cervical tissue samples and keratinocytes. Eight of these genes (CDKN2A, ELAVL2, GRB7, HSPB1, KHSRP, NOVA1, PTBP1, and RNASEH2A) encode RNA-binding proteins. Further studies indicated that both HPV16 and HPV18 infections lead to the aberrant expression of selected RBP-encoding genes. We found that viral E6 and E7 decrease NOVA1 expression but that E7 increases RNASEH2A expression via E2F1. The altered expression of these genes may be utilized as biomarkers for high-risk (HR)-HPV carcinogenesis and progression.

DNA damage and reactive oxygen species cause cell death in the rice local lesions 1 mutant under high light and high temperature.

High light and high temperature (HLHT) stress may become more frequent and severe as the climate changes, affecting crop growth and resulting in reduced production. However, the mechanism of the response to HLHT stress in rice is not yet fully understood. In the present study, we screened a rice mutant library using HLHT conditions and isolated an HLHT-sensitive mutant, local lesions 1 (ls1), which showed decreased pigment contents, defective stomata and chloroplasts, and a local lesions phenotype under HLHT. We characterized and cloned LS1 by map-based cloning and genetic complementation. LS1 encodes the A subunit of the RNase H2 complex (RNASEH2A). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and comet assays indicated that mutation of LS1 led to severe DNA damage under HLHT stress. Furthermore, we found excessive reactive oxygen species (ROS) accumulation in the ls1 mutant under HLHT stress. Exogenous antioxidants eased the local lesions phenotype of the ls1 mutant under HLHT. DNA damage caused by HLHT stress induces ROS accumulation, which causes the injury and apoptosis of leaf cells in the ls1 mutant. These results enhance our understanding of the regulatory mechanism in the response to HLHT stress in higher plants.

Prognostic Value of RNASEH2A-, CDK1-, and CD151-Related Pathway Gene Profiling for Kidney Cancers.

The nucleotide degrading enzyme gene RNASEH2A (ribonuclease H2 subunit A) has been found to be overexpressed in cancers. Our aim was to understand the role of RNASEH2A in cancer prognostication and to establish a scoring system based on the expressions of genes interacting with RNASEH2A. We screened the nucleotide degrading enzyme gene expression in RNAseq data of 14 cancer types derived from The Cancer Genome Atlas (TCGA) and found that RNASEH2A overexpression was associated with poor patient survival only in renal cell carcinomas (RCCs). Further cluster analyses of samples with poor outcomes revealed that cluster of differentiation 151 (CD151) upregulation correlated with low cyclin dependent kinase 1 (CDK1) and high RNASEH2A expression. The combination of low CD151 expression and high RNASEH2A expression resulted in impaired proliferation in four kidney cancer cell lines, suggesting potential synthetic dosage lethality (SDL) interactions between the two genes. A prognostication scoring system was established based on the expression levels of RNASEH2A-, CDK1-, and CD151-related genes, which could effectively predict the overall survival in a TCGA clear cell RCC cohort (n = 533, 995.3 versus 2242.2 days, p < 0.0001), in another clear cell renal cell carcinoma (ccRCC) cohort E-GEOD-22541 (n = 44, 390.0 versus 1889.2 days, p = 0.0007), and in a TCGA papillary RCC (pRCC) cohort (n = 287, 741.6 versus 1623.7 days, p < 0.0001). Our results provide a clinically applicable prognostication scoring system for renal cancers.

Clinical and genetic analysis in a patient with type 4 Aicardi-Goutières syndrome / 临床儿科杂志

Objective To explore the clinical characteristics, imaging and genetic features of Type 4 Aicardi-Goutières syndrome (AGS). Methods The clinical data were collected, genetic changes were tested using next generation sequencing, and relevant literatures were reviewed. Results A 5 months old girl present with recurrent fever, intelligence and motor developmental delay, epilepsy, microcephaly, spasticity, cerebrospinal fluid pleocytosis. Brain MRI displayed cerebral atrophy and white matter lesions. Brain CT displayed intra-cranial multiple calcifications. Two missense mutations were identified in RNASEH2A,c.199G>C was a novel mutation,and c.322C>T was a known pathogenic mutation.Conclusions RNASEH2A gene mutations can lead to type 4 AGS.

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families.