Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

Effects of transgenic cotton lines expressing dsAgCYP6CY3-P1 on the growth and detoxification ability of Aphis gossypii glover.

BACKGROUND: The pest Aphis gossypii Glover globally causes considerable economic losses on various crops by its feeding damage and disease transmission. Transgenic plants that produce double-stranded RNA (dsRNA) targeted to insect genes are being developed as a pest control strategy. In this study, we evaluated the effects of transgenic cotton-mediated RNA interference (RNAi) on the growth and detoxification ability of A. gossypii after the transgenic cotton lines expressing dsAgCYP6CY3-P1 (the TG cotton lines) were obtained on the basis of exploring the functions of CYP6CY3 in our previous research. RESULTS: The developmental time of third- and fourth-instar nymphs which fed on the TG cotton lines were significantly prolonged. Life table parameters showed that the fitness of cotton aphids from the TG cotton lines decreased. Additionally, the relative expression level of CYP6CY3 in cotton aphids which fed on the TG cotton lines was significantly reduced by 47.3 % at 48 h compared with that from the nontransgenic cotton (the NT cotton). Bioassay showed that silencing of CYP6CY3 increased mortality of the nymphs to imidacloprid by 28.49 % (at 24 h) and to acetamiprid by 73.77 % (at 48 h), respectively. CONCLUSION: These results indicated that the TG cotton lines delayed the growth and development of A. gossypii, but also decreased population density and increased its sensitivity to imidacloprid and acetamiprid, respectively. The results provide further support for the development and application of plant-mediated RNAi. © 2022 Society of Chemical Industry.

Melatonin Prevents NaAsO2-Induced Developmental Cardiotoxicity in Zebrafish through Regulating Oxidative Stress and Apoptosis.

Melatonin is an indoleamine hormone secreted by the pineal gland. It has antioxidation and anti-apoptosis effects and a clear protective effect against cardiovascular diseases. Our previous studies demonstrated that embryonic exposure to sodium arsenite (NaAsO2) can lead to an abnormal cardiac development. The aim of this study was to determine whether melatonin could protect against NaAsO2-induced generation of reactive oxygen species (ROS), oxidative stress, apoptosis, and abnormal cardiac development in a zebrafish (Danio rerio) model. We found that melatonin decreased NaAsO2-induced zebrafish embryonic heart malformations and abnormal heart rates at a melatonin concentration as low as 10-9 mol/L. The NaAsO2-induced oxidative stress was counteracted by melatonin supplementation. Melatonin blunted the NaAsO2-induced overproduction of ROS, the upregulation of oxidative stress-related genes (sod2, cat, gpx, nrf2, ho-1), and the production of antioxidant enzymes (Total SOD, SOD1, SOD2, CAT). Melatonin attenuated the NaAsO2-induced oxidative damage, DNA damage, and apoptosis, based on malonaldehyde and 8-OHdG levels and apoptosis-related gene expression (caspase-3, bax, bcl-2), respectively. Melatonin also maintained the control levels of heart development-related genes (nkx2.5, sox9b) affected by NaAsO2. In conclusion, melatonin protected against NaAsO2-induced heart malformations by inhibiting the oxidative stress and apoptosis in zebrafish.

The Zinc Finger Antiviral Protein ZAP Restricts Human Cytomegalovirus and Selectively Binds and Destabilizes Viral UL4/UL5 Transcripts.

Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a ß-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized UL4-UL6 HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the UL4-UL6 locus may play an important role for HCMV replication.IMPORTANCE Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.

Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering.

Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

Identifying transposable element expression dynamics and heterogeneity during development at the single-cell level with a processing pipeline scTE.

Transposable elements (TEs) make up a majority of a typical eukaryote's genome, and contribute to cell heterogeneity in unclear ways. Single-cell sequencing technologies are powerful tools to explore cells, however analysis is typically gene-centric and TE expression has not been addressed. Here, we develop a single-cell TE processing pipeline, scTE, and report the expression of TEs in single cells in a range of biological contexts. Specific TE types are expressed in subpopulations of embryonic stem cells and are dynamically regulated during pluripotency reprogramming, differentiation, and embryogenesis. Unexpectedly, TEs are expressed in somatic cells, including human disease-specific TEs that are undetectable in bulk analyses. Finally, we apply scTE to single-cell ATAC-seq data, and demonstrate that scTE can discriminate cell type using chromatin accessibly of TEs alone. Overall, our results classify the dynamic patterns of TEs in single cells and their contributions to cell heterogeneity.

The SARS-CoV-2 RNA-protein interactome in infected human cells.

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae.

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Ammonium Polyphosphate with High Specific Surface Area by Assembling Zeolite Imidazole Framework in EVA Resin: Significant Mechanical Properties, Migration Resistance, and Flame Retardancy.

A zeolite imidazole framework (ZIF-67) was assembled onto the surface of ammonium polyphosphate (APP) for preparing a series multifunctional flame-retardant APP-ZIFs. The assembly mechanism, chemical structure, chemical compositions, morphology, and specific surface area of APP-ZIFs were characterized. The typical APPZ1 and APPZ4 were selected as intumescent flame retardants with dipentaerythritol (DPER) because of their superior unit catalytic efficiency of cobalt by thermogravimetric analysis. APPZ1 and APPZ4 possessed 6.8 and 92.1 times the specific surface area of untreated APP, which could significantly enhance the interfacial interaction, mechanical properties, and migration resistance when using in ethylene-vinyl acetate (EVA). With 25% loading, 25% APPZ4/DPER achieved a limiting oxygen index value of 29.4% and a UL 94 V-0 rating, whereas 25% APP/DPER achieved a limiting oxygen index value of only 26.2% and a V-2 rating, respectively. The peak of the heat release rate, smoke production rate, and CO production rate respectively decreased by 34.7%, 39.0%, and 40.1%, while the char residue increased by 91.7%. These significant improvements were attributed to the catalytic graphitization by nano cobalt phosphate and the formation of a more protective char barrier comprised of graphite-like carbon.

Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2.

Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

DNA-mediated dimerization on a compact sequence signature controls enhancer engagement and regulation by FOXA1.

FOXA1 is a transcription factor capable to bind silenced chromatin to direct context-dependent cell fate conversion. Here, we demonstrate that a compact palindromic DNA element (termed 'DIV' for its diverging half-sites) induces the homodimerization of FOXA1 with strongly positive cooperativity. Alternative structural models are consistent with either an indirect DNA-mediated cooperativity or a direct protein-protein interaction. The cooperative homodimer formation is strictly constrained by precise half-site spacing. Re-analysis of chromatin immunoprecipitation sequencing data indicates that the DIV is effectively targeted by FOXA1 in the context of chromatin. Reporter assays show that FOXA1-dependent transcriptional activity declines when homodimeric binding is disrupted. In response to phosphatidylinositol-3 kinase inhibition DIV sites pre-bound by FOXA1 such as at the PVT1/MYC locus exhibit a strong increase in accessibility suggesting a role of the DIV configuration in the chromatin closed-open dynamics. Moreover, several disease-associated single nucleotide polymorphisms map to DIV elements and show allelic differences in FOXA1 homodimerization, reporter gene expression and are annotated as quantitative trait loci. This includes the rs541455835 variant at the MAPT locus encoding the Tau protein associated with Parkinson's disease. Collectively, the DIV guides chromatin engagement and regulation by FOXA1 and its perturbation could be linked to disease etiologies.

Facile synthesis of boron- and nitride-doped MoS2 nanosheets as fluorescent probes for the ultrafast, sensitive, and label-free detection of Hg(2+).

Bulk MoS2, a prototypical transition metal chalcogenide material, is an indirect band gap semiconductor with negligible photoluminescence. In this study, we have developed, for the first time, a simple and low-cost synthetic strategy to prepare boron- and nitrogen-doped MoS2 (B,N-MoS2) nanosheets. Through boron and nitrogen doping, the band gap of MoS2 increases from 1.20 eV to 1.61 eV, and the obtained B,N-MoS2 nanosheets exhibit an enhanced fluorescence. The B,N-MoS2 nanosheets can be used as a green and facile sensing platform for label-free detection of Hg(2+) because of their high sensitivity and selectivity toward Hg(2+). In addition, detection can be easily accomplished through one-step rapid (within 2 min) operation, with a limit as low as 1 nM. This study demonstrates that the introduction of boron and nitrogen elements into ultrathin MoS2 nanosheets for enhanced fluorescence properties is feasible through a facile and general preparation strategy and may also offer a unique idea as a potential way to design more efficient MoS2-based sensors and fluorescent materials.