Ever-Expanding NGLY1 biology

The cytosolic peptide:N-glycanase (PNGase; NGLY1 in humans) is a deglycosylating enzyme that is widely conserved in eukaryotes. This enzyme is involved in the degradation of misfolded N-glycoproteins that are destined for proteasomal degradation in the cytosol, a process that is called endoplasmic reticulum (ER)-associated degradation (ERAD). Although the physiological significance of NGLY1 remained unknown until recently, the discovery of NGLY1 deficiency, a human genetic disorder bearing mutations in the NGLY1 gene, has led to explosive research progress regarding the functional characterization of this enzyme. For example, it is now known that NGLY1 can also act as an “editing enzyme” to convert N-glycosylated asparagine residues to aspartate residues, thus introducing negative charges into a core peptide and modulating the function of the target molecule. Diverse biological processes have also been found to be affected by compromised NGLY1 activity. In this special issue, recent research progress on the functional characterization of NGLY1 and its orthologues in worm/fly/rodents, assay methods/biomarkers useful for the development of therapeutics, and the comprehensive transcriptome/proteome of NGLY1-KO cells as well as patient-derived cells are discussed.

ADAR1 RNA editing regulates endothelial cell functions via the MDA-5 RNA sensing signaling pathway

The RNA-sensing signaling pathway has been well studied as an essential antiviral mechanism of innate immunity. However, its role in non-infected cells is yet to be thoroughly characterized. Here, we demonstrated that the RNA sensing signaling pathway also reacts to the endogenous cellular RNAs in endothelial cells (ECs), and this reaction is regulated by the RNA-editing enzyme ADAR1. Cellular RNA sequencing analysis showed that EC RNAs endure extensive RNA editing, especially in the RNA transcripts of short interspersed nuclear elements. The EC-specific deletion of ADAR1 dramatically reduced the editing level on short interspersed nuclear element RNAs, resulting in newborn death in mice with damage evident in multiple organs. Genome-wide gene expression analysis revealed a prominent innate immune activation with a dramatically elevated expression of interferon-stimulated genes. However, blocking the RNA sensing signaling pathway by deletion of the cellular RNA receptor MDA-5 prevented interferon-stimulated gene expression and rescued the newborn mice from death. This evidence demonstrated that the RNA-editing/RNA-sensing signaling pathway dramatically modulates EC function, representing a novel molecular mechanism for the regulation of EC functions.

Downregulated ADARB1 Facilitates Cell Proliferation, Invasion and has Effect on the Immune Regulation in Ovarian Cancer

Ovarian cancer (OC) is typically diagnosed at an advanced stage and poses a significant challenge to treatment and recovery. Rencently, Adenosine deaminase RNA-specific B1 (ADARB1), an adenosine-to-inosine (A-to-I) RNA-editing enzyme, has been found to play an essential role in the development of cancer. However, the specific function of ADARB1 in ovarian cancer is still not fully understood. Here, we investigated the effects of ADARB1 on OC biology. By conducting bioinformatics analyses of several public databases, we found significantly decreased ADARB1 expression in OC cells and tissues. Moreover, RT-PCR and western blot showed lower ADARB1 expression in OVCAR3, HO8910pm and A2780 OC cells compared to human normal ovarian epithelial cell IOSE. Cell proliferation assay and clone formation assay showed that overexpression of ADARB1 (ADARB1-OE) inhibited the proliferation of tumor cells. Wound healing and transwell assay indicated that ADARB1-OE could suppress OC cell invasion and metastasis. Kaplan-Meier methods revealed that the patients with low level of ADARB1 displayed poor prognosis. TISIDB databases were further used to analyze the roles of ADARB1 in tumor-immune system interactions in OC patients. Furthermore, ADARB1-OE down-regulated the expression of phosphorylated AKT. Combination of ADARB1-OE and AKT inhibitor MK2206 exerted stronger cell growth inhibition. Thus, our investigation demonstrated that low levels of ADARB1 might be a potential target in the tumorigenesis and prognostic evaluation of OC patients.

Conservation of A-to-I RNA editing in bowhead whale and pig

RNA editing is a post-transcriptional process in which nucleotide changes are introduced into an RNA sequence, many of which can contribute to proteomic sequence variation. The most common type of RNA editing, contributing to nearly 99% of all editing events in RNA, is A-to-I (adenosine-to-inosine) editing mediated by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes. A-to-I editing at ‘recoding’ sites results in non-synonymous substitutions in protein-coding sequences. Here, we present studies of the conservation of A-to-I editing in selected mRNAs between pigs, bowhead whales, humans and two shark species. All examined mRNAs–NEIL1, COG3, GRIA2, FLNA, FLNB, IGFBP7, AZIN1, BLCAP, GLI1, SON, HTR2C and ADAR2 –showed conservation of A-to-I editing of recoding sites. In addition, novel editing sites were identified in NEIL1 and GLI1 in bowhead whales. The A-to-I editing site of human NEIL1 in position 242 was conserved in the bowhead and porcine homologues. A novel editing site was discovered in Tyr244. Differential editing was detected at the two adenosines in the NEIL1 242 codon in both pig and bowhead NEIL1 mRNAs in various tissues and organs. No conservation of editing of KCNB1 and EEF1A mRNAs was seen in bowhead whales. In silico analyses revealed conservation of five adenosines in ADAR2, some of which are subject to A-to-I editing in bowheads and pigs, and conservation of a regulatory sequence in GRIA2 mRNA that is responsible for recognition of the ADAR editing enzyme.

The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, off-target mutagenesis by A3A has been readily detected in both cellular DNA and RNA, which has been shown to promote oncogenesis. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA, but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G) which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

Emerging Severe Acute Respiratory Syndrome Coronavirus 2 Mutation Hotspots Associated With Clinical Outcomes and Transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding the influence of mutations in the SARS-CoV-2 gene on clinical outcomes is critical for treatment and prevention. Here, we analyzed all high-coverage complete SARS-CoV-2 sequences from GISAID database from January 1, 2020, to January 1, 2021, to mine the mutation hotspots associated with clinical outcome and developed a model to predict the clinical outcome in different epidemic strains. Exploring the cause of mutation based on RNA-dependent RNA polymerase (RdRp) and RNA-editing enzyme, mutation was more likely to occur in severe and mild cases than in asymptomatic cases, especially A > G, C > T, and G > A mutations. The mutations associated with asymptomatic outcome were mainly in open reading frame 1ab (ORF1ab) and N genes; especially R6997P and V30L mutations occurred together and were correlated with asymptomatic outcome with high prevalence. D614G, Q57H, and S194L mutations were correlated with mild and severe outcome with high prevalence. Interestingly, the single-nucleotide variant (SNV) frequency was higher with high percentage of nt14408 mutation in RdRp in severe cases. The expression of ADAR and APOBEC was associated with clinical outcome. The model has shown that the asymptomatic percentage has increased over time, while there is high symptomatic percentage in Alpha, Beta, and Gamma. These findings suggest that mutation in the SARS-CoV-2 genome may have a direct association with clinical outcomes and pandemic. Our result and model are helpful to predict the prevalence of epidemic strains and to further study the mechanism of mutation causing severe disease.

ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

A20 Inhibits LPS-Induced Inflammation by Regulating TRAF6 Polyubiquitination in Rainbow Trout

The ubiquitin-editing enzyme A20 is known to inhibit the NF-κB transcription factor in the Toll-like receptor (TLR) pathways, thereby negatively regulating inflammation. However, its role in the TLR signaling pathway in fish is still largely unknown. Here, we identified a gene encoding A20 (OmA20) in rainbow trout, Oncorhynchus mykiss, and investigated its role in TLR response regulation. The deduced amino acid sequence of OmA20 contained a conserved N-terminal ovarian tumor (OTU) domain and seven C-terminal zinc-finger (ZnF) domains. Lipopolysaccharide (LPS) stimulation increased OmA20 expression in RTH-149 cells. In LPS-stimulated RTH-149 cells, gain- and loss-of-function experiments revealed that OmA20 inhibited MAPK and NF-κB activation, as well as the expression of pro-inflammatory cytokines. OmA20 interacted with TRAF6, a key molecule involved in the activation of TLR-mediated NF-κB signaling pathways. LPS treatment increased the K63-linked polyubiquitination of TRAF6 in RTH-149 cells, which was suppressed when OmA20 was forced expression. Furthermore, mutations in the OTU domain significantly decreased deubiquitination of the K63-linked ubiquitin chain on TRAF6, indicating that deubiquitinase activity is dependent on the OTU domain. These findings suggest that OmA20, like those of mammals, reduces LPS-induced inflammation in rainbow trout, most likely by regulating K63-linked ubiquitination of TRAF6.