Selective translational control of cellular and viral mRNAs by RPS3 mRNA binding.

RPS3, a universal core component of the 40S ribosomal subunit, interacts with mRNA at the entry channel. Whether RPS3 mRNA-binding contributes to specific mRNA translation and ribosome specialization in mammalian cells is unknown. Here we mutated RPS3 mRNA-contacting residues R116, R146 and K148 and report their impact on cellular and viral translation. R116D weakened cap-proximal initiation and promoted leaky scanning, while R146D had the opposite effect. Additionally, R146D and K148D displayed contrasting effects on start-codon fidelity. Translatome analysis uncovered common differentially translated genes of which the downregulated set bears long 5'UTR and weak AUG context, suggesting a stabilizing role during scanning and AUG selection. We identified an RPS3-dependent regulatory sequence (RPS3RS) in the sub-genomic 5'UTR of SARS-CoV-2 consisting of a CUG initiation codon and a downstream element that is also the viral transcription regulatory sequence (TRS). Furthermore, RPS3 mRNA-binding residues are essential for SARS-CoV-2 NSP1-mediated inhibition of host translation and for its ribosomal binding. Intriguingly, NSP1-induced mRNA degradation was also reduced in R116D cells, indicating that mRNA decay occurs in the ribosome context. Thus, RPS3 mRNA-binding residues have multiple translation regulatory functions and are exploited by SARS-CoV-2 in various ways to influence host and viral mRNA translation and stability.

Translation regulation of specific mRNAs by RPS26 C-terminal RNA-binding tail integrates energy metabolism and AMPK-mTOR signaling.

Increasing evidence suggests that ribosome composition and modifications contribute to translation control. Whether direct mRNA binding by ribosomal proteins regulates the translation of specific mRNA and contributes to ribosome specialization has been poorly investigated. Here, we used CRISPR-Cas9 to mutate the RPS26 C-terminus (RPS26dC) predicted to bind AUG upstream nucleotides at the exit channel. RPS26 binding to positions -10 to -16 of short 5' untranslated region (5'UTR) mRNAs exerts positive and negative effects on translation directed by Kozak and Translation Initiator of Short 5'UTR (TISU), respectively. Consistent with that, shortening the 5'UTR from 16 to 10 nt diminished Kozak and enhanced TISU-driven translation. As TISU is resistant and Kozak is sensitive to energy stress, we examined stress responses and found that the RPS26dC mutation confers resistance to glucose starvation and mTOR inhibition. Furthermore, the basal mTOR activity is reduced while AMP-activated protein kinase is activated in RPS26dC cells, mirroring energy-deprived wild-type (WT) cells. Likewise, the translatome of RPS26dC cells is correlated to glucose-starved WT cells. Our findings uncover the central roles of RPS26 C-terminal RNA binding in energy metabolism, in the translation of mRNAs bearing specific features and in the translation tolerance of TISU genes to energy stress.

Selecting for CRISPR-Edited Knock-In Cells.

CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, "scarless" selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.

CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is "scarless" since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system's utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.

A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1.

NAD(P)H:quinone-oxidoreductase-1 (NQO1) is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. NQO1 has been also shown to rescue proteins containing intrinsically unstructured domains, such as p53 and p73, from degradation by the 20S proteasome through an unknown mechanism. Here, we studied the nature of interaction between NQO1 and the 20S proteasome. Our study revealed a double negative feedback loop between NQO1 and the 20S proteasome, whereby NQO1 prevents the proteolytic activity of the 20S proteasome and the 20S proteasome degrades the apo form of NQO1. Furthermore, we demonstrate, both in vivo and in vitro, that NQO1 levels are highly dependent on FAD concentration. These observations suggest a link between 20S proteolysis and the metabolic cellular state. More generally, the results may represent a regulatory mechanism by which associated cofactors dictate the stability of proteins, thus coordinating protein levels with the metabolic status.

The nonreceptor tyrosine kinase c-Src attenuates SCF(ß-TrCP) E3-ligase activity abrogating Taz proteasomal degradation.

The polyomavirus middle T antigen (PyMT) oncogene activates the cellular nonreceptor tyrosine kinase c-Src and recruits the Hippo pathway effectors, Yap (yes-associated protein) and Taz (transcriptional coactivator with PDZ-binding motif), as key steps in oncogenesis. Yap and Taz are transcription coactivators shuttling from the cytoplasm to the nucleus. The Hippo pathway kinase Lats1/2 (large tumor suppressor homolog) reduces Yap/Taz nuclear localization and minimizes their cytoplasmic levels by facilitating their ubiquitination by the E3 ligase SCF(ß-TrCP). In contrast, PyMT increases the cytoplasmic Taz level. Here we show that this unique PyMT behavior is mediated by Src. We demonstrate that PyMT-induced Src activation inhibits degradation of both wild-type and tyrosine-less Taz, ruling out Taz modification as a mechanism of escaping degradation. Instead, we found that Src attenuates the SCF(ß-TrCP) E3-ligase activity in blunting Taz proteasomal degradation. The role of Src in rescuing Taz from TrCP-mediated degradation gives rise to higher cell proliferation under dense cell culture. Finally, IkB (NF-kappa-B inhibitor), a known substrate of ß-TrCP, was rescued by Src, suggesting a wider effect of Src on ß-TrCP substrates. These findings introduce the Src tyrosine kinase as a regulator of SCF(ß-TrCP).

Recruitment of the protein phosphatase-1 catalytic subunit to promoters by the dual-function transcription factor RFX1.

RFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes. The exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes.

The Disordered Landscape of the 20S Proteasome Substrates Reveals Tight Association with Phase Separated Granules.

Proteasomal degradation is the main route of regulated proteostasis. The 20S proteasome is the core particle (CP) responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. It has been previously shown that the 20S CP is active in degradation of certain intrinsically disordered proteins (IDP) lacking structural constrains. Here, a comprehensive analysis of the 20S CP substrates in vitro is conducted. It is revealed that the 20S CP substrates are highly disordered. However, not all the IDPs are 20S CP substrates. The group of the IDPs that are 20S CP substrates, termed 20S-IDPome are characterized by having significantly more protein binding partners, more posttranslational modification sites, and are highly enriched for RNA binding proteins. The vast majority of them are involved in splicing, mRNA processing, and translation. Remarkably, it is found that low complexity proteins with prion-like domain (PrLD), which interact with GR or PR di-peptide repeats, are the most preferential 20S CP substrates. The finding suggests roles of the 20S CP in gene transcription and formation of phase-separated granules.

c-Abl tyrosine kinase promotes adipocyte differentiation by targeting PPAR-gamma 2.

Adipocyte differentiation, or adipogenesis, is a complex and highly regulated process. A recent proteomic analysis has predicted that the nonreceptor tyrosine kinase Abelson murine leukemia viral oncogene (c-Abl) is a putative key regulator of adipogenesis, but the underlying mechanism remained obscure. We found that c-Abl was activated during the early phase of mouse 3T3-L1 preadipocyte differentiation. Moreover, c-Abl activity was essential and its inhibition blocked differentiation to mature adipocytes. c-Abl directly controlled the expression and activity of the master adipogenic regulator peroxisome proliferator-activator receptor gamma 2 (PPARγ2). PPARγ2 physically associated with c-Abl and underwent phosphorylation on two tyrosine residues within its regulatory activation function 1 (AF1) domain. We demonstrated that this process positively regulates PPARγ2 stability and adipogenesis. Remarkably, c-Abl binding to PPARγ2 required the Pro12 residue that has a phenotypically well-studied common human genetic proline 12 alanine substitution (Pro12Ala) polymorphism. Our findings establish a critical role for c-Abl in adipocyte differentiation and explain the behavior of the known Pro12Ala polymorphism.

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage.

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.

NADH binds and stabilizes the 26S proteasomes independent of ATP.

The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.

Hepatitis B virus induces RNR-R2 expression via DNA damage response activation.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infects and replicates in quiescent hepatocytes, which are deficient in dNTPs, the critical precursors of HBV replication. Most tumor viruses promote dNTP production in host cells by inducing cell proliferation. Although HBV is known as a major cause of hepatocellular carcinoma, it does not lead to cellular proliferation. Instead, HBV acquires dNTPs by activating the expression of the R2 subunit of the Ribonucleotide Reductase (RNR) holoenzyme, the cell cycle gene that is rate-limiting for generation of dNTPs, without inducing the cell cycle. We wished to elucidate the molecular basis of HBV-dependent R2 expression in quiescent cells. METHODS: Quiescent HepG2 cells were transduced with an HBV-containing lentiviral vector, and primary human hepatocytes were infected with HBV. DNA damage response and RNR-R2 gene expression were monitored under this condition. RESULTS: We report here that HBV-induced R2 expression is mediated by the E2F1 transcription factor, and that HBV induces E2F1 accumulation, modification and binding to the R2 promoter. We found that Chk1, a known E2F1 kinase that functions in response to DNA damage, was activated by HBV. In cells where Chk1 was pharmacologically inhibited, or depleted by shRNA-mediated knockdown, HBV-mediated R2 expression was severely attenuated. Furthermore, we found that HBV attenuates DNA repair, thus reducing cellular dNTP consumption. CONCLUSIONS: Our findings demonstrate that HBV exploits the Chk1-E2F1 axis of the DNA damage response pathway to induce R2 expression in a cell cycle-independent manner. This suggests that inhibition of this pathway may have a therapeutic value for HBV carriers.

BIM(EL), an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation.

BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase ß-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

Depleting the 19S proteasome regulatory PSMD1 subunit as a cancer therapy strategy.

BACKGROUND: Proteasome inhibitors are in use in treating certain types of cancers. These drugs inhibit the catalytic activity of the 20S proteasome, shared by all the different proteasome complexes. Inhibitors of the 26S-associated deubiquitinating activity explicitly inhibit the 26S proteasomal degradation of ubiquitinylated substrates. We have previously reported an alternative strategy that is based on reducing the 26S/20S ratio by depleting PSMD1, 6, and 11, the subunits of the 19S proteasome regulatory complex. Given the addiction of the many cancer types to a high 26S/20S ratio, the depletion strategy is highly effective in killing many aggressive cancer cell lines but not mouse and human immortalized and normal cells. METHODS: We used two aggressive cell lines, MDA-MB-231, a triple-negative breast tumor cell line, and OVCAR8, a high-grade ovary adenocarcinoma. Cell culture, mouse MDA-MB-231, OVCAR8 xenografts, and patient-derived ovarian cancer xenograft (PDX) models were transduced with lentivectors expressing PSMD1 shRNA. Tumor size was measured to follow treatment efficacy. RESULTS: Using different experimental strategies of expressing shRNA, we found that PSMD1 depletion, either by expressing PSMD1 shRNA in an inducible manner or in a constitutive manner, robustly inhibited MDA-MB-231, and OVCAR8 xenograft tumor growth. Furthermore, the PSMD1 depletion strategy compromised the growth of the PDX of primary ovarian cancer. CONCLUSION: Our results suggest that reducing the 26S/20S ratio might be a valuable strategy for treating drug-resistant aggressive types of cancers.

Method of Monitoring 26S Proteasome in Cells Revealed the Crucial Role of PSMA3 C-Terminus in 26S Integrity.

Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multi-subunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generates the 26S proteasome, which is the major proteasomal complex in cells. Native gel protocols are used to investigate the 26S/20S ratio. However, a simple method for detecting these proteasome complexes in cells is missing. To this end, using CRISPR technology, we YFP-tagged the endogenous PSMB6 (ß1) gene, a 20S CP subunit, and co-tagged endogenous PSMD6 (Rpn7), a 19S RP subunit, with the mScarlet fluorescent protein. We observed the colocalization of the YFP and mScarlet fluorescent proteins in the cells, with higher nuclear accumulation. Nuclear proteasomal granules are formed under osmotic stress, and all were positive for YFP and mScarlet. Previously, we have reported that PSMD1 knockdown, one of the 19 RP subunits, gives rise to a high level of "free" 20S CPs. Intriguingly, under this condition, the 20S-YFP remained nuclear, whereas the PSMD6-mScarlet was mostly in cytoplasm, demonstrating the distinct subcellular distribution of uncapped 20S CPs. Lately, we have shown that the PSMA3 (α7) C-terminus, a 20S CP subunit, binds multiple intrinsically disordered proteins (IDPs). Remarkably, the truncation of the PSMA3 C-terminus is phenotypically reminiscent of PSMD1 knockdown. These data suggest that the PSMA3 C-terminal region is critical for 26S proteasome integrity.

The Transmembrane Protease Serine 2 (TMPRSS2) Non-Protease Domains Regulating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike-Mediated Virus Entry.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Via the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus transduction, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2's cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.

SARS-CoV-2 Omicron Specific Mutations Affecting Infectivity, Fusogenicity, and Partial TMPRSS2-Independency.

The COVID-19 pandemic resulted from the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since its first appearance in 2019, new SARS-CoV-2 variants of concern (VOCs) have emerged frequently, changing the infection's dynamic. SARS-CoV-2 infects cells via two distinct entry routes; receptor-mediated endocytosis or membrane fusion, depending on the absence or presence of transmembrane serine protease 2 (TMPRSS2), respectively. In laboratory conditions, the Omicron SARS-CoV-2 strain inefficiently infects cells predominantly via endocytosis and is phenotypically characterized by decreased syncytia formation compared to the earlier Delta variant. Thus, it is important to characterize Omicron's unique mutations and their phenotypic manifestations. Here, by utilizing SARS-CoV-2 pseudovirions, we report that the specific Omicron Spike F375 residue decreases infectivity, and its conversion to the Delta S375 sequence significantly increases Omicron infectivity. Further, we identified that residue Y655 decreases Omicron's TMPRSS2 dependency and entry via membrane fusion. The Y655H, K764N, K856N and K969N Omicron revertant mutations, bearing the Delta variant sequence, increased the cytopathic effect of cell-cell fusion, suggesting these Omicron-specific residues reduced the severity of SARS-CoV-2. This study of the correlation of the mutational profile with the phenotypic outcome should sensitize our alertness towards emerging VOCs.

Cholesterol 24-hydroxylase at the choroid plexus contributes to brain immune homeostasis.

The choroid plexus (CP) plays a key role in remotely controlling brain function in health, aging, and disease. Here, we report that CP epithelial cells express the brain-specific cholesterol 24-hydroxylase (CYP46A1) and that its levels are decreased under different mouse and human brain conditions, including amyloidosis, aging, and SARS-CoV-2 infection. Using primary mouse CP cell cultures, we demonstrate that the enzymatic product of CYP46A1, 24(S)-hydroxycholesterol, downregulates inflammatory transcriptomic signatures within the CP, found here to be elevated across multiple neurological conditions. In vitro, the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) downregulates CYP46A1 expression, while overexpression of CYP46A1 or its pharmacological activation in mouse CP organ cultures increases resilience to TNF-α. In vivo, overexpression of CYP46A1 in the CP in transgenic mice with amyloidosis is associated with better cognitive performance and decreased brain inflammation. Our findings suggest that CYP46A1 expression in the CP impacts the role of this niche as a guardian of brain immune homeostasis.

E3 ligase STUB1/CHIP regulates NAD(P)H:quinone oxidoreductase 1 (NQO1) accumulation in aged brain, a process impaired in certain Alzheimer disease patients.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that is important in maintaining the cellular redox state and regulating protein degradation. The NQO1 polymorphism C609T has been associated with increased susceptibility to various age-related pathologies. We show here that NQO1 protein level is regulated by the E3 ligase STUB1/CHIP (C terminus of Hsc70-interacting protein). NQO1 binds STUB1 via the Hsc70-interacting domain (tetratricopeptide repeat domain) and undergoes ubiquitination and degradation. We demonstrate here that the product of the C609T polymorphism (P187S) is a stronger STUB1 interactor with increased susceptibility to ubiquitination by the E3 ligase STUB1. Furthermore, age-dependent decrease of STUB1 correlates with increased NQO1 accumulation. Remarkably, examination of hippocampi from Alzheimer disease patients revealed that in half of the cases examined the NQO1 protein level was undetectable due to C609T polymorphism, suggesting that the age-dependent accumulation of NQO1 is impaired in certain Alzheimer disease patients.

Evidence for a Hepatitis B Virus Short RNA Fragment Directly Targeting the Cellular RRM2 Gene.

The hepatitis B virus (HBV) is one of the smallest but most highly infectious human pathogens. With a DNA genome of only 3.2 kb and only four genes, HBV successfully completes its life cycle by using intricate processes to hijack the host machinery. HBV infects non-dividing liver cells in which dNTPs are limited. As a DNA virus, HBV requires dNTPs for its replication. HBV induces the ATR-mediated cellular DNA damage response pathway to overcome this constraint. This pathway upregulates R2 (RRM2) expression in generating an active RNR holoenzyme catalyzing de novo dNTP synthesis. Previously we reported that ERE, a small RNA fragment within the HBx ORF, is sufficient to induce R2 upregulation. Interestingly, there is high sequence similarity between ERE and a region within the R2 5'UTR that we named R2-box. Here, we established a mutant cell line in the R2-box region of the R2 gene using CRISPR-Cas9 technology to investigate the R2 regulation by ERE. This cell line expresses a much lower R2 level than the parental cell line. Interestingly, the HBV infection and life cycle were severely impaired. These cells became permissive to HBV infection upon ectopically R2 expression. These results validate the requirement of the R2 gene expression for HBV replication. Remarkably, the R2-box mutated cells became ERE refractory, suggesting that the homology region between ERE and R2 gene is critical for ERE-mediated R2 upregulation. Thus, along with the induction of the ATR pathway of the DNA damage response, ERE might also directly target the R2 gene via the R2-box.