To understand SARS-CoV-2 evolution and vaccine efficacy, it is critical to identify the extent and distribution of errors introduced by the RNAPs involved in each process. Current methods lack the sensitivity and specificity to measure de novo RNA variants in low input samples like viral isolates3. Here, we determine the frequency and nature of RNA errors in both SARS-CoV-2 and its vaccine using a targeted Accurate RNA Consensus sequencing method (tARC-seq). We found that the viral RNA-dependent RNAP (RdRp) makes ~1 error every 10,000 nucleotides – higher than previous estimates4. We also observed that RNA variants are not randomly distributed across the genome but are associated with certain genomic features and genes, such as S (Spike). tARC-seq captured a number of large insertions, deletions and complex mutations that can be modeled through non-programmed RdRp template switching. This template switching feature of RdRp explains many key genetic changes observed during the evolution of different lineages worldwide, including Omicron. Further sequencing of the Pfizer-BioNTech COVID-19 vaccine revealed an RNA variant frequency of ~1 in 5,000, meaning most of the vaccine transcripts produced in vitro by T7 phage RNAP harbor a variant. These results demonstrate the extraordinary genetic diversity of viral populations and the heterogeneous nature of an mRNA vaccine fueled by RNAP inaccuracy. Along with functional studies and pandemic data, tARC-seq variant spectra can inform models to predict how SARS-CoV-2 may evolve. Finally, our results may help improve future vaccine development and study design as mRNA therapies continue to gain traction.Both the SARS-CoV-2 virus and its mRNA vaccines depend on RNA polymerases (RNAP) Both SARS-CoV-2 and the mRNA vaccines rely on viral RNA polymerases (RNAPs) for their respective replication or synthesis. RNAPs misincorporate nucleotides at much higher frequencies than their DNA counterparts3, generating RNA variants during synthesis which can fuel viral evolution. The mutation frequency of RNA viruses ranges from 10−4 to 10−6 per base7. SARS-CoV-2 is commonly believed to acquire mutations more slowly than other RNA viruses due to the proofreading activity of its exonuclease during RNA synthesis8. However, to this point, no empirical studies have directly measured the frequency of RdRp errors during replication  with the potential to escape vaccine protection. mRNA vaccines based on the Spike protein have been widely used to prevent COVID-19 illness and have been shown to elicit a protective immune response against VOCs after multiple doseslication , which ilication .10. While these advances in consensus sequencing have reduced technical noise, they typically require considerable substrate (≥1 μg of RNA) and are not feasible for low input samples11. Another major constraint is that variant discovery is directly correlated with sequencing depth, and depth can be difficult to achieve for rare transcripts or organisms with large and complex genomes. To overcome these limitations, we developed targeted Accurate RNA Consensus sequencing (tARC-seq)  pose a major challenge to RNA variant detection. To remove these artifacts, new methods that combine RNA-seq with aspects of circle sequencing and molecular barcoding were developed (ARC-seq) to enable accurate variant callingARC-seq) . tARC-seEscherichia coli, where hybrid capture produced a >30-fold enrichment on average in unique consensus reads across a panel of twelve genes , followed by deletions (2.49E-5) and insertions (3.22E-6). Nearly 70% of point mutations were nonsynonymous , additional studies are needed.Applying tARC-seq to the B.1.1.7 isolate (Alpha), we measured an RNA variant frequency of 1.51E-4 . SignifiPosition-wise calculations in Alpha again revealed a number of hot and cold spots for RNA variants . Genes e5 to 106 pfu/mL), tARC-seq produced fewer consensus reads for Delta. While read depth was sufficient for genome-wide studies, positional variant frequencies are not reported as the tests were underpowered.The B.1.617.2 isolate (Delta) had a similar RNA variant frequency (1.43E-4) as Alpha . DespiteAltogether, ultra-deep sequencing across three different lineages has revealed a relatively high error frequency for SARS-CoV-2’s RdRp, despite having proofreading activity.17. This programmed template switching is driven by sequence complementarity between TRS sequences, and it functions to add a common leader sequence to viral transcripts to enhance gene expression18. tARC-seq detected fusion transcripts in WT SARS-CoV-2 with junctions mapping to canonical TRS sites  to a leader sequence (TRS-L) in the 5’ UTR to generate subgenomic mRNAsRS sites . MoreoveRS sites –d.19. Analyzing tARC-seq data, we observe many recurrent junctions outside canonical TRSs in fusion transcripts  by T7 phage polymerase from a codon-optimized, Spike-encoding DNA construct   , we char−4, or double that of WT SARS-CoV-2  compared to the vaccine template, implicating low GC content in template switching  the native S gene from WT SARS-CoV-2, and (2) the codon-optimized Spike construct from the Pfizer vaccine. The two templates differ significantly by nucleotide content, with the modified vaccine template being 57% GC compared to 37% for the viral template. mRNA from either IVT reaction had a reduced variant frequency compared to the vaccine , but difwitching . We alsowitching , corrobowitching –b.25, mRNA purification, liposomal packaging, vaccine freezing and storage, or the scale up for production, also contribute to the RNA variant frequencies observed in the vaccine samples..After controlling for GC enrichment and standard T7 IVT conditions, we conclude that the intrinsic error-prone nature of T7 RNAP is the main source of vaccine variants. However, other features specific to Pfizer vaccine production, such as the use of N1-methylpseudouridine4, this frequency is comparable to similar observations in poliovirus, which also utilizes an RdRp for replication but lacks an associated proofreading activity26. The error frequency estimations were previously based on the presence of a proofreading 3’-to-5’ exoribonuclease  in SARS-CoV-2 that is distinct from the viral RdRp27. The same proofreading activity has been implicated in promoting template switching28, which we show here is error-prone. Thus, our work highlights the promiscuous nature of SARS-CoV-2’s RdRp driven by nucleotide misincorporation and erroneous template switching, both controlled by the same exonuclease. ExoN may be a key protein involved in tuning viral evolution. Together, these results showcase the fundamental biology propelling viral diversity and evolution on a massive scale during the COVID-19 pandemic.Herein, we describe a targeted sequencing method for detecting RNA variants in rare transcripts and low abundance samples. We have sequenced three SARS-CoV-2 isolates and established a baseline variant frequency of ~1 in 10,000 per nucleotide for the virus. While higher than other predictions31. Vaccine variants could promote a more diverse immune repertoire, which offers benefits in the context of a rapidly evolving virus. However, other uses like cancer vaccines or mRNA drugs may require high fidelity transcription to reduce the risk of autoimmunity or improve clinical efficacy2. Identification and usage of high fidelity RNAP may be crucial for the development of future therapies. Importantly, our results build on a growing body of work promoting mRNA-based drug technologies in medicine and public health.In conjunction with viral data, we also measured RNA variants in the Pfizer BioNTech COVID-19 vaccine using ARC-seq. At a frequency of 1 in 5,000 nucleotides, the pace of vaccine variants appears balanced against viral evolution and suggests that the majority of mRNA produced encodes variant Spike proteins. The role of vaccine heterogenicity in the immune response is currently unknown. Our data may provide insight to explain how mRNA vaccines against COVID-19 offer broader protection against novel strains upon boostingFinally, beyond the COVID-19 pandemic, tARC-seq has revealed many new principles concerning basic RdRp biology. RNA errors are non-random and linked to GC content, transcriptional patterns and sequence complementarity. RdRp is capable of non-programmed template switching to form structural variants, insertions and deletions, ultimately fueling virus evolution. Future research should expound on the sequence motifs and rules that regulate RNA errors and polymerase switching events as they likely afflict every corner of life. Perhaps then we can begin to understand how organisms have evolved to mitigate RNAP infidelity or learned to exploit it.9. RNA integrity was confirmed via Agilent Tapestation™ prior to sequencing library preparation.All RNA samples were processed under RNase-free conditions using dedicated equipment and reagents. To maintain RNA integrity, samples were limited to ≤1 freeze-thaw cycle, kept on ice whenever possible, and not subjected to high temperatures (≥65 C) in the presence of metal cations 33. Briefly, 48-72 hours post infection, supernatant from infected cultures were collected and a plaque assay was performed using Vero E6 cells to calculate viral titer. Virus was inactivated in TRIzol™ reagent before freezing at −80 C for long-term storage. RNA was exacted from thawed TRIzol preps 34 at the time of library preparation.Ancestral SARS-CoV-2 was received from the World Reference Center for Emergeing Viruses and Arboviruses at The University of Texas Medical Branch  under the direction of Drs. Scott Weaver and Kenneth Plante. Alpha variant  and Delta variant  were received from BEI Resources. Viral stocks were prepared by infecting Vero CCL-81 cells as previously describedVaccine was collected from the remains of unexpired, spent vials  that had been stored at room temperature for <6 hours, as per the manufacturer’s recommendations for proper handling. No vaccine that could otherwise have been administered to patients was sacrificed for the purposes of this study. Vaccine samples were transferred on wet ice. Upon receipt, RNA was immediately extracted with PhenokChloroformTsoamyl Alcohol (25:24:1) (PCA) followed by ethanol precipitation. Samples were reconstituted in nuclease-free water and analyzed by Qubit Fluorometer™ and Agilent Tapestation™ prior to storage at −80 C.35. Following harvest, sample cleanup was performed with the RNA Clean & Concentrator Kit (Zymo Research) and DNase treatment (TURBO™ DNase) was applied off-column at 37 C for 1 hour. The ribosomal RNA fraction was depleted via the RiboMinus™ Transcriptome Isolation Kit for bacteria (Invitrogen™), and the resulting mRNA was concentrated by ethanol precipitation for downstream library preparation.Luria broth was inoculated from isolated colonies and grown for 16 hours at 37 C. The next day, overnight cultures were washed, diluted 100-fold in fresh LB, and grown at 37 to mid-log phase (OD600 ~0.4). 1 mL culture aliquots were then harvested in duplicate and placed on ice preceding RNA extraction using the RNAsnap™ protocolin vitro transcription (IVT): (I) the native SARS-CoV-2 Spike gene (Addgene 145670), and (II) the codon-optimized, GC-enriched spike-encoding construct from Pfizer-BioNTech 37. Both genes were expressed from bacterial expression plasmids downstream of T7 promoters. Plasmids were isolated from E. coli cultures using the QIAprep Spin Miniprep Kit™ and subjected to single-enzyme digest with SnaBI  or Mlul (vaccine template). Linearized plasmid was then cleaned up by PCA and verified by Qubit Fluorometer™ and gel electrophoresis before IVT. Reactions were performed in parallel at escalating temperatures  using the Hi Scribe® T7 High Yield RNA Synthesis Kit (NEB) and the ProFlex PCR System Thermal Cycler (Applied Biosystems). Starting from 200 ng of template, reactions were allowed to proceed for two hours before TURBO™ DNase treatment. Transcription products were purified by PCA for sequencing. Notably, while IVT from the vaccine template was less efficient and had lower yields, vaccine transcripts were more resistant to RNaseIII fragmentation during library preparation.Two different templates were used for 38. Following PCA cleanup, the remaining steps of library construction were guided by ARC-seq design 10. Briefly, RNA was 5’ adenylated and ligated to unique molecular identifiers (UMIs). Barcoding individual fragments before any amplification reactions downstream is critical for error correction during consensus calling. The library was then circularized and primed for rolling-circle reverse-transcription, yielding multiple conjoined copies of the original fragment. After digesting these long cDNA oligomers, the individual monomers were PCR amplified with tailed primers to add sequencing adapters and additional indexes. The final library was analyzed by Tapestation prior to paired-end sequencing on the Illumina NextSeq 550 system. Importantly, reaction conditions were optimized throughout to reduce RNA damage from heat and metal ion catalysis.1 μg of RNA was enzymatically fragmented with RNaseIII for 7 min to an average size of 450 nt E. coli sample was mixed 4:1 with viral RNA and served as both the carrier and internal control. Once fragmented, the targeted libraries are prepared by the total RNA protocol up through the last indexing step. SARS-CoV-2 reads were then enriched using the COVID xGen Hybrid Capture Kit (IDT) and 7-9 cycles of post-capture PCR to generate the final library.For concentrated samples (≥ 100 ng/μL), fragmentation can proceed as described above. However, for low-abundance samples it is recommended to add carrier RNA up to 1 μg. For the SARS-CoV-2 experiments, a previously sequenced Illumina BCL files were converted to Fastq and demultiplexed from the 6 nt sample barcode in the i7 index read . Next, UMIs were extracted and appended to the read headers before converting to unaligned BAM format. Specifically, the 11 nt PCR UMI was read from the i5 index, and the 16 nt cDNA UMI was clipped from the leading 16 bases of Read 2. Reads with identical UMI tags, originating from the same RNA fragment, were grouped into families and collapsed into consensus reads using a custom python script. Consensus FASTQ sequences were aligned to the appropriate reference genome using BWA-MEM . Reads were then quality filtered, clipped, and realigned with GATK v3.8. Finally, variants were called using mpileup and tabulated via a custom R script. (Scheme S1)E. coli hybrid capture was performed on 2 biological replicates. Viral analysis was performed on 1 biological replicate per genotype. Vaccine sequencing data represents 2 vials of the same lot (n=2). In vitro experiments were performed in triplicate at 30, 37, and 42 C (n=1 per temperature).Frequencies were calculated as variant count / total consensus nucleotides sequenced. Proportion confidence intervals (two-tailed) were calculated for each frequency and error bars represent Wilson scores of 95% confidence. To detect positions in the SARS-CoV-2 genome that have a base substitution frequency different from the sample average, we constructed a contingency table and performed Fisher’s exact test comparing each position to the genome-wide average. All positions that passed our initial filter  were included in this analysis. To control the false discovery rate, p-values were adjusted using the Benjamini–Hochberg procedure. Positions with adjusted p-values <0.05 and substitution frequencies that are elevated or reduced compared to the genome-wide average were called as hot and cold spots, respectively. 39 was then colored based on the codon average RNA base substitution frequency using Pymol 40.Average RNA base substitution frequencies were calculated for each codon in the Spike protein by sum(base substitution count)/sum(adjusted depth). All positions that passed our initial filter  and were sequenced at a raw depth ≥10000 were included in this analysis. The Spike protein full-length model 6vsb_1_1_1


Their genome has two open reading frames (ORFs) that encode capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). The toti-like viruses recently identified in Anopheles sp. and Aedes aegypti mosquitoes (AaTV) share the same genome organization as other totiviruses. The AaTVs that have been described in distinct geographical regions are monophyletic. In this study, we show that AaTV sequences can be grouped into at least three phylogenetic clades . Clades A and B are composed of AaTV sequences from mosquitoes collected in the Caribbean region (Guadeloupe), and clade C contains sequences from the USA. These clades may represent AaTV lineages that are locally adapted to their host populations. We also identified three recombinant AaTV strains circulating in mosquitoes in Guadeloupe. Although these strains have different chimeric patterns, the position of the recombination breakpoint was identical in all strains. Interestingly, this breakpoint is located in a hairpin-like structure in the intergenic region of the AaTV genome. This RNA structure may stall RNA polymerase processivity and consequently induce template switching. In vitro studies should be conducted to further investigate the biological significance of AaTV’s intergenic region as a recombination hotspotThe genus  Totiviridae contains five genera  [Totivirus contains viruses initially identified in fungi [Salmo salmo) [Anopheles and Aedes aegypti) share the genomic characteristics described for Totiviridae and share an ancestral monophyletic ancestor [Totiviridae family and have not been definitively classified. Likewise, Guadeloupe Aedes aegypti totivirus (AaTV) is an insect-specific virus that was identified in Aedes aegypti from the Caribbean [The family rivirus) ,2. The gin fungi . More toin fungi ,5,6,7. Tin fungi ,10,11,12in fungi , as wellin fungi   . The totancestor ,17,18. Taribbean ,20,21. Taribbean .Aedes aegypti totivirus (AaTV). We found three sequences with similar mosaic patterns. They also share the location of the single breakpoint detected in the intergenic region of the AaTV. We also hypothesized that a predicted secondary RNA structure found in the intergenic region of AaTV may halt the processivity of RNA-polymerase. This can induce the RNA-polymerase to detach and jump to another RNA target template, thus increasing the chances of genomic recombination.In a previous study, the authors speculated about a possible recombination event, but the results were inconclusive . Here, wAedes aegypti totivirus (AaTV) from NCBI . We also included in this dataset one Totivirus sequence detected in Fungi (MZ868719), and Totiviruses sequences of other insects . We downloaded all 25 near-complete genomes of The alignment was performed using MAFFT  and edithttp://darwin.uvigo.es/rdp/rdp.html/ accessed on 22 September 2022). We used RDP v.5 software , which uhttp://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi/ accessed on 22 September 2022). RNAfold predicts the minimum free energy (mfe) structure of a single sequence using the classic algorithm of Zuker and Stiegler, which uses thermodynamics and auxiliary information. In addition, the method calculates equilibrium base pairing probabilities via John McCaskill’s partition function algorithm.To predict the secondary RNA structure, we used the RNAfold method of the Vienna RNA web services (available at Aedes aegypti (AaTVs) and one totivirus (LC496074) identified in Anopheles gambiae . Thus, we were interested in determining the extent to which varying substitution rates affected the clade composition detected in our previous analysis. To minimize the effect of rate heterogeneity in the inferences, we used the GTR model plus gamma correction and estimated the proportion of invariable sites. Both capsid and RdRp trees showed similar topological structures, presenting three AaTV clades . In addiy-axis in x-axis in y-axis in y-axis in The phylogenetic analysis showed that the sequences MN053724, MN053725, and MN053732 were in different clades in trees constructed with capsid and RdRp, and these findings suggest that these sequences have parts of their genomes composed of distinct segments originating from different phylogenetic clades. Thus, we decided to initially detect any signal of recombination in the alignment composed of near-complete AaTV sequences. AaTV MT435499 was excluded from the recombination analysis owing to the poor quality of this sequence. We used an approach (INTERVAL) that estimates site-by-site variations in recombination rates along the lengths of nucleotide sequence alignments. This can therefore potentially be used in a similar way to identify the recombination breakpoint density signal. The recombination rates  to predict the secondary RNA structure. The predicted structure shows a hairpin-like structure that is formed before position 2933  of totiviruses is very structured and functions as an internal ribosome entry site (IRES). The noncoding intergenic region is highly conserved among AaTVs; it is also prone to the formation of a structure. It is plausible that the RNA structure predicted in the intergenic region of AaTV may affect the processivity of the RNA polymerase during replication. Thus, by stalling RNA polymerase, the RNA structure facilitates template switching and the synthesis of chimeric AaTV genomes. There is much empirical evidence that RNA secondary structures induce homologous recombination in viruses ,47. For Mosquitoes harbor a great variety of human pathogenic viruses, and they function as reservoirs of potential new pathogens. Here, we provide some examples of how AaTV can increase genomic diversity through recombination that forms chimeric genomes composed of genes from distinct clades. Thus, the role of insect viruses in the emergence of new arboviruses requires further investigation.


High cholesterol-induced bone loss is highly associated with oxidative stress, which leads to the generation of oxysterols, such as 7-ketocholesterol (7-KC). Here, we conducted in vivo and in vitro experiments to determine whether arctiin prevents high cholesterol diet-induced bone loss by decreasing oxidative stress. First, arctiin was orally administered to atherogenic diet (AD)-fed C57BL/6J male mice at a dose of 10 mg/kg for 6 weeks. Micro-computerized tomography (μCT) analysis showed that arctiin attenuated AD-induced boss loss. For our in vitro experiments, the anti-oxidant effects of arctiin were evaluated in 7-KC-stimulated osteoclasts (OCs). Arctiin decreased the number and activity of OCs and inhibited autophagy by disrupting the nuclear localization of transcription factor EB (TFEB) and downregulating the oxidized TFEB signaling pathway in OCs upon 7-KC stimulation. Furthermore, arctiin decreased the levels of reactive oxygen species (ROS) by enhancing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), catalase, and heme oxygenase 1 (HO-1), all of which affected OC differentiation. Conversely, silencing of Nrf2 or HO-1/catalase attenuated the effects of arctiin on OCs. Collectively, our findings suggested that arctiin attenuates 7-KC-induced osteoclastogenesis by increasing the expression of ROS scavenging genes in the Nrf2/HO-1/catalase signaling pathway, thereby decreasing OC autophagy. Moreover, arctiin inhibits the oxidation and nuclear localization of TFEB, thus protecting mice from AD-induced bone loss. Our findings thus demonstrate the therapeutic potential of arctiin for the prevention of cholesterol-induced bone loss. Emerging evidence has suggested a positive correlation between high cholesterol levels and decreased bone density ,2,3. In Arctium lappa L. that has long been used as a medicinal herb in East Asia [Arctiin, an arctigenin glycoside, is a bioactive ingredient of ast Asia . Specifiast Asia . In miceast Asia . Moreoveast Asia . Arctiinast Asia . After oast Asia . Arctigeast Asia . Additioast Asia . Arctiinast Asia .Based on the aforementioned findings, we hypothesized that arctiin may prevent high cholesterol diet-induced bone loss by decreasing oxidative stress. Therefore, we investigated the effects of arctiin on bone mass in atherogenic diet-fed mice in vivo and its molecular mechanisms in osteoclasts (OCs) upon 7-KC stimulation in vitro.2DCFDA), and HRP-conjugated secondary antibodies were acquired from Thermo Fisher Scientific . Antibodies against lamin B1 (#13435) and microtubule-associated proteins 1A/1B light chain 3B  were provided by Cell Signaling . Antibodies against β-actin (A5441) and transcription factor EB  were purchased from Sigma Chemical and Bethyl Laboratories Inc , respectively. The scrambled siRNA , nuclear factor erythroid 2-related factor 2 (Nrf2) small interfering RNA (siRNA) , heme oxygenase 1 (HO-1) siRNA , catalase siRNA , peroxiredoxin (PRDX) 1 siRNA , PRDX4 siRNA , and horseradish peroxidase (HRP)-streptavidin were provided by Santa Cruz Biotechnology .Arctiin was obtained from Chengdu Herb Purify Co., Ltd. . Macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) were purchased from R&D Systems, Inc. . Opti-MEM and M-MLV reverse transcriptase were provided by Gibco  and Promega , respectively. 7-KC, a leukocyte acid phosphatase (TRAP) kit, N-Acetyl-L-cysteine (NAC), MTT -2,5-diphenyltetrazolium bromide), diphenyleneiodonium chloride (DPI), and toluidine blue were provided by Sigma Chemical . SYBR Green was purchased from Qiagen . The OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit (Colorimetric) was purchased from Cell Biolabs Inc. . The RatLaps EIA kit was obtained from Immunodiagnostic Systems Inc. . The TRIzol reagent, lipofectamine RNAiMAX transfection reagent, and N-(Biotinoyl)-N’-(iodoacetyl) ethylenediamine (BIAM) were acquired from Invitrogen . An alkaline phosphatase (ALP) colorimetric kinetic assay and an osteocalcin EIA kit were purchased from BioAssay Systems  and Biomedical Technologies Inc. , respectively. The SYBR Green Real-Time PCR Master Mixes, NE-PER nuclear and cytoplasmic extraction reagents, 2′,7′-dichlorofluorescein diacetate (Hn = 4), AD + PBS (n = 4), and AD + arctiin . Arctiin was dissolved in dimethyl sulfoxide (DMSO) and adjusted to a 0.5% DMSO concentration by diluting it with PBS; the mice were orally treated with different concentrations of arctiin (or just PBS as a vehicle control) every day for 6 weeks. For micro-computed tomography (μCT) and serum extraction, the animals were randomly divided into the following four groups: ND + PBS (n = 5), ND + arctiin (n = 5), AD + PBS (n = 5), and AD + arctiin (n = 5). The mice were orally treated with 10 mg/kg of arctiin or PBS every day for 6 weeks. All mice were kept in a specific pathogen-free environment. All standards employed herein were approved by the UOUACUC (HSC-21-010). In addition, all animal care and operations were performed in accordance with protocols and guidelines accepted by the Animal Care and Use Committee at the University of Ulsan (UOUACUC). After 6 weeks, in order to acquire serum, the mice were anesthetized, and a heparinized capillary tube was used to retro-orbitally collect a blood sample. Then, the mice were euthanized with CO2 and the femurs were harvested for X-ray radiogram or μCT analysis. For radiographic analysis, mouse femora were cleaned and stored in 10% formalin for a week. Femur radiographs were taken by soft X ray  according to the manufacturers’ instructions. Relative intensities of the distal femurs from X-ray radiograms were measured by using the ImageJ (version 1.37) software. To visualize long bone architecture, femurs were analyzed by scanning with a high-resolution μCT imaging system in a SkyScan 1072 System  set to a 6.9 μm effective detector pixel size and a 77–255 mg/cc threshold. Trabecular bone was analyzed in a 1.7 mm region located 0.4 mm below the distal femur growth plate. Three-dimensional analyses were performed with the CT volume software  using a total of 75–125 tomographic slices. To examine in vivo TRAP-positive OCs, mouse femora were excised, and decalcified in EDTA. Representative histologic sections of the distal femoral metaphysis of mice fed with AD or ND in the presence or absence of arctiin were stained for TRAP and hematoxylin and eosin (H&E) to identify OCs  and growth plates , respectively. Serum C-telopeptide fragments of collagen type 1 (CTX-1) were measured as an in vivo marker of bone resorption using the RatLaps enzyme immunoassay (EIA). Serum ALP and osteocalcin were measured with a colorimetric kinetic assay and an osteocalcin EIA kit, respectively. Serum H2O2 was measured with the OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit (Colorimetric). All experiments were carried out according to the manufacturers’ instructions.The C57BL/6J male mice, ten-weeks-old, were obtained from The Jackson Laboratory  and the mice were fed with either a chow  or atherogenic diet (AD) for 6 weeks. The mouse AD was provided by Research Diets, Inc. . For X-ray radiogram, the mice were randomly divided into five groups: ND + phosphate-buffered saline (PBS)  for 48 h. Then, M-CSF (30 ng/mL) and RANKL (40 ng/mL) were added to the BMMs, and the cells were fixed in 10% formalin for 10 min after incubation for the times indicated in each figure legend. The cells were then stained for tartrate-resistant alkaline phosphatase (TRAP) as described in a previous study [Bone marrow cells were obtained from six-week-old C57BL/6J male mice as previously described . To obtaus study . The areus study . The num® iD3 multi-mode microplate reader .The effect of arctiin on cell viability was determined by MTT assay. Briefly, the BMMs were treated with different concentrations of arctiin  in the presence of M-CSF (30 ng/mL), RANKL (40 ng/mL) and 7-KC (7 μM) until mature osteoclasts were formed. Then, the cells were treated with 0.5 mg/mL MTT ) at 37 °C for 30 min. After removing the medium, DMSO (80 μL) was added to the cells and the absorbance was measured at 540 nm using a SpectraMax4OH and the dentine slices were stained with 1% (w/v) toluidine blue in 0.5% sodium borate to visualize the resorption pits. The resorption pit area and TRAP-positive cells were measured with the ImageJ (version 1.37) software.To assess the effect of arctiin on the function of OCs, we further implanted mature OCs on dentine slices for 4 to 5 days as previously described . In shor−∆∆Ct method was used to calculate the copy numbers relative to RPS expression.To synthesize single-stranded cDNA, total RNA was extracted and reverse-transcribed using M-MLV reverse transcriptase and random primers. QPCR was performed using SYBR Green  with the appropriate primers. The specific primers used are shown in To evaluate the localization of TFEB and the expression of LC3B, the cell proteins were extracted by using NE-PER nuclear and cytoplasmic extraction reagents and radioimmune precipitation lysis buffer, respectively. SDS-PAGE was used to separate the protein, which was then transferred to a nitrocellulose membrane. After that, the membranes were blocked for 1 h at room temperature with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (1X TBS-T). Next, the specific primary antibodies against LC3B (1:1000), β-actin , TFEB (1:1000), and lamin B1 (1:1000) were incubated at 4 °C for overnight with shaking, then the membranes were washed with 1X TBS-T and incubated with secondary antibodies for 1 h. Finally, the membranes were developed using chemiluminescent substrates after being washed three times with 1X TBS-T. The intensity of the bands was determined using ImageJ (version 1.37) software.To investigate the effect of arctiin on autolysosomes, flow cytometry was utilized to analyze acidic vesicular organelles  after staining with acridine orange (AO), as previously described . AO is a2DCFDA) was used to determine intracellular ROS. In brief, BMMs were cultured with M-CSF (30 ng/mL), RANKL (40 ng/mL), and 7-KC (7 μM) in the presence of different concentrations of arctiin  for 48 h. The cells were then washed twice with PBS and treated with fresh α-MEM containing 10 μM H2DCFDA and incubated at 37 °C in the dark for 10–15 min. Intracellular ROS were detected by using a FACSCalibur flow cytometer .The fluorescent probe 2′,7′-dichlorofluorescein diacetate (HBMMs were treated with 7-KC (7 μM) ± arctiin (15 μM) in the presence of M-CSF (30 ng/mL) and RANKL (40 ng/mL) for 51 h. The cells were then harvested and flash-frozen in liquid nitrogen. Next, the frozen cells were transferred to a lysis buffer containing 100 μM BIAM and the solution was deoxygenated for 20 min by bubbling nitrogen gas at a low flow rate. BIAM is extensively employed as a sulfhydryl modifying agent that selectively detects the reduced form of cysteine . The extBMMs were treated with M-CSF (30 ng/mL), RANKL (40 ng/mL), and 7-KC (7 μM) in the presence or absence of arctiin (15 μM) for 3 h and then transfected with siRNA against Nrf2 or HO-1 and catalase or with scRNA as a negative control using the lipofectamine RNAiMAX transfection reagent according to the manufacturer’s instructions. After transfection, the cells were cultured with M-CSF (30 ng/mL), RANKL (40 ng/mL), and 7-KC (7 μM) with or without arctiin (15 μM) for the times indicated in the figure legends.t-test. One-way ANOVA was used to evaluate group differences followed by Bonferroni post-hoc tests. A statistically significant difference was considered as a p-value < 0.05.All data were presented as means ± standard deviation (SD). The differences between the samples and the corresponding controls were assessed by using Student’s To investigate whether arctiin prevented cholesterol-rich AD-induced bone loss, mice fed with either a high cholesterol diet  or an isocaloric low-cholesterol control diet  for 6 weeks were orally treated with arctiin or vehicle (PBS). A 10 mg/kg dose of arctiin treatment exhibited maximum protection compared to doses of 5 mg/kg and 30 mg/kg, as demonstrated by X-ray radiogram analyses . MoreoveOur previous data suggested that increased dietary cholesterol decreased BMD  with enhOur previous results revealed that 7-KC enhanced autophagy by facilitating TFEB nuclear localization in OCs . TherefoWe next sought to investigate the molecular mechanisms through which arctiin affected OCs. In our previous study, we demonstrated that the stimulatory effects of 7-KC on OCs were due to its capacity to increase ROS levels . TherefoArctium lappa [Our findings demonstrated that arctiin, an arctigenin glycoside purified from um lappa , protectum lappa . Our in um lappa . In addium lappa ,26. Howeum lappa . In our Our previous study demonstrated that AD-induced bone loss was accompanied by oxidative stress as well as increased 7-KC levels in bone , suggestCollectively, our findings, at least partly, demonstrated that arctiin up-regulates the expression of Nrf2 and its target genes including HO-1 and catalase, thereby decreasing ROS levels in OCs and inhibiting cholesterol-induced bone loss. Furthermore, arctiin abrogates autophagy by disrupting 7-KC-stimulated TFEB nuclear localization via decreasing oxidation in OCs . Therefo


Cytidine and adenosine deaminases are required for cytosine and adenine editing of base editors respectively, and no single deaminase could enable concurrent and comparable cytosine and adenine editing. Additionally, distinct properties of cytidine and adenosine deaminases lead to various types of off-target effects, including Cas9-indendepent DNA off-target effects for cytosine base editors (CBEs) and RNA off-target effects particularly severe for adenine base editors (ABEs). Here we demonstrate that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single or double mutations, which display minimized Cas9-independent DNA off-target effects and genotoxicity, with orthologs B5ZCW4, Q57LE3, E8WVH3, Q13XZ4 and B3PCY2 as promising candidates for further engineering. Furthermore, RNA off-target effects of TadA ortholog-derived base editors could be further reduced or even eliminated by additional single mutation. Taken together, our work expands the base editing toolkits, and also provides important clues for the potential evolutionary process of deaminases. Properties of cytidine and adenosine deaminases lead to off-target effects for cytosine base editors (CBEs) and adenine base editors (ABEs). Here the authors report that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single/double mutations with minimised off-targets. Escherichia coli (hereafter ecTadA for wild-type version and ecTadA7.10 for evolved version in ABE7.10) to edit adenine(s) and CBEs containing cytidine deaminases to edit cytosine(s) at target genomic regions5. Cytidine and adenosine deaminases also display distinct properties leading to various types of off-target effects10. For example, ABEs displayed obvious RNA off-target effects as wild-type ecTadA is an RNA-specific deaminase, while CBEs displayed significant Cas9-independent DNA off-target effects due to intrinsic single-stranded DNA (ssDNA) affinity of cytidine deaminases17. As off-target effects severely restricted the applications of base editors, deaminase engineering and screening have been extensively exploited to improve base editing tools24. Nevertheless, deaminases with optimized properties remain limited, and thus constrained the editing outcomes of base editors. Moreover, as no single deaminase enables comparable cytosine and adenine editing simultaneously, ACBEs require two distinct deaminases fusing together, which leads to a larger size to restrict their utility27.Distinct deaminases are required for base editors to induce cytosine or adenine editing, with ABEs containing evolved tRNA-specific adenosine deaminases from 31. Therefore, TadA deaminases represented promising candidates for the generation of ABEs, CBEs, and ACBEs. However, it is well-known that wild-type ecTadA deaminase could not edit adenines at the DNA level, and more than ten amino acid substitutions, identified by directed protein evolution, were required for ecTadA engineering to generate potent ABEs2. In consideration of the complicated ecTadA evolution process, additional TadA orthologs have not been exploited and engineered for functional base editors.It has been demonstrated that ABEs could catalyze cytosine deamination within specific sequence contexts, and particular mutations could modulate the editing preference for adenine or cytosineHere, we show that in combined with an internal fusion strategy, functional base editors, including ABEs, CBEs, and ACBEs, are generated with various TadA orthologs containing just one or two amino acid substitutions. Additionally, RNA off-target effects are further minimized by single amino acid substitution. This study provides an alternative and simplified strategy for orthologous TadA screening and engineering, which would be valuable for the expansion and diversification of base editing tools, and also provide insights into the potential evolutionary process of deaminases.18, we examined whether domain insertion strategy could also enhance the editing capacity of ecTadA(VN)-derived ABEs. In addition to the conventional N-terminus site, ten representative insertion sites inside nCas9, including docking site (DS) 535/583/770/793/801/895/905/919/1029/1249, were selected for ecTadA(VN) insertion and editing capacity was evaluated at a specific endogenous site containing multiple targetable cytosines and adenines (targeting by sgRNA-1) in HEK293T cells. In consistent with previous studies, N-terminal derived N-ecTadA(VN) displayed marginal editing capacity ) for functional ABEs to achieve adenine editing at the DNA level. However, ecTadA(VN) displayed marginal adenine editing and additional complicated engineering was required to enhance its editing capacity. As ecTadA7.10 insertion inside nCas9 could enhance the editing capacity and modulate substrate specificity of ABEsity Fig.  while seity Fig. . To exclity Fig. . It was ity Fig. , with N-ity Fig. . In cons32 , Salmonella choleraesuis TadA (Q57LE3), and Streptococcus pyogenes TadA (Q5XE14)), were chosen for protein engineering and screening (Uniprot ID was used for orthologs nomenclature). Generally, only residues corresponding to A106 and D108 of ecTadA were substituted with V and N in TadA orthologs (detailed protein sequences in Supplementary Data As ecTadA(VN) fusing inside DS1249 of nCas9 enabled functional ABEs with only A106V and D108N double mutations, we wondered whether other TadA orthologs could also be simply engineered with corresponding double mutations to generate functional ABEs, CBEs, and ACBEs without complicated directed protein evolution. About 1000 TadA orthologs described previously34. Here we revealed that TadA-8e and ecTadA(VN)-derived ABEs also induced significantly increased γH2AX accumulation  as compared to nCas9 (average of 12.2%), while 6/9 representative TadA ortholog-derived base editors displayed similar γH2AX  to nCas9 , we evaluated the RNA off-target effects of several representatives TadA ortholog-derived functional base editors. It was revealed that derived functional base editors, regardless of ABEs, ACBEs, or CBEs, all induced detectable A-to-I (average of ~2.5–6.6-fold) but no obvious C-to-U RNA off-target edits as compared to nCas9 Fig. , indicatas9 Fig. . TherefoWe subsequently selected six representatives TadA ortholog-derived base editors 1249-NL-B5ZCW4(VN), E8WVH3(VN), Q13XZ4(VN), B3PCY2(VN), Q57LE3(VN), and Q99W51(VN), for editing signature analysis at multiple endogenous sites with high-throughput sequencing. Additionally, 1249-NL-ecTadA7.10 and -ecTadA(VN) were also included as controls. 1249-NL-ecTadA7.10, with ecTadA7.10 as a significantly improved version, mainly served as ABE and displayed editing scope across A4-A12 with robust editing activity (>60% A-to-G conversion frequency), while 1249-NL-ecTadA(VN) displayed editing scope at A5 with modest editing activity (~40% A-to-G conversion frequency) Fig. . 1249-NLCC > CG) and AC, 1249-NL-Q13XZ4(VN) preferred CW (CA/CT > CC/CG) and WC (AC/TC > CC/GC) while 1249-NL-B3PCY2(VN) preferred CT/AC and displayed minimal cytosine editing activity at CG/GC contexts  preferred CW 23, or V82G/W (simplified as VG or VW)12, could significantly reduce or even eliminate RNA off-target edits of ecTadA-derived ABEs. Here we introduced similar mutations into representative 1249-NL-Q13XZ4(VN), -B3PCY2(VN), -E8WVH3(VN), -Q57LE3(VN), and -Q99W51(VN), which displayed obvious A-to-I RNA off-target edits, and evaluated the changes of editing activity and RNA off-target edits at specific sites. It was revealed that Q13XZ4(VN) (FA), B3PCY2(VN) (Rdel), E8WVH3(VN) (FA), and Q57LE3(VN) (Rdel) significantly reduced A-to-I RNA off-target edits at the selected site while maintaining comparable on-target editing activities ies Fig. . We furties Fig. .Fig. 4Mi2. Here we demonstrated that in combined with an internal fusion strategy at specific insertion sites of nCas9, engineering of ecTadA with just two amino acid substitutions at conserved residues could generate functional ABEs with obvious adenine editing activity and detectable cytosine editing activity, consistent with previous studies showing that classical ABEs containing evolved ecTadA displayed cytosine editing activity within specific sequence contexts31. By screening tens of different TadA orthologs in HEK293T cells with flow cytometry  was initially considered as ABE in our deaminase screening against five endogenous sites Fig. , while t37. Considering that the engineering of TadA orthologs enabled the generation of functional ABEs, CBEs, and ACBEs in this study, adenosine deaminases TadAs represented alternative strategies to develop optimized CBEs and miniCBEs. In the future, systematic exploration of TadA orthologs, combined with critical amino acids identified during the TadA-8e reprogramming process, would guide the generation of conventional or miniature base editors with diversified editing signatures to expand current toolkits.We demonstrated in another study published simultaneously that ecTadA variant TadA-8e could be reprogrammed to generate potent CBEs and ACBEs with minimized DNA/RNA off-target edits. Additionally, reprogrammed TadA-8e variants enabled the generation of potent miniCBEs with high precision and minimized off-target effects2. Nevertheless, cytidine and adenosine deaminases have a similar active pocket and highly conserved catalytic H(C)XE and PCXXC motifs, indicating an evolutionary relationship between them38. Recent studies have also demonstrated that evolved TadA variant TadA-8e could be engineered to enable robust C-to-T editing with or without A-to-G editing capacity31, further supporting a potential evolutionary relationship between cytidine and adenosine deaminases. However, a single natural protein possessing both C-U and A-I conversion activities has not been identified, and only one enzymatic complex, TbADAT2/3 from Trypanosoma brucei, has been reported to mediate both C-U deamination in ssDNA and A-I in tRNA in vitro39. In consideration of the engineering results of TadA orthologs, we constructed a simplified phylogenetic tree for adenosine and cytidine deaminases with respective deamination capacity against different substrates . The mutagenesis strategy  was used to induce specific mutations for corresponding base editors. To generate TadA ortholog-derived base editors, the docking site at residue 1249 was initially inserted with SpeI-NLS-BamHI-NheI-linker-XbaI sequence, and then engineered orthologous TadAs were inserted via BamHI-NheI double digestion strategy. Plasmids expressing dSaCas9-UGI-T2A-mCherry and U6-sgsaRNA, which were used in R-loop assay, were derived from PX602 (Addgene #61593), in which D10A and N580A were induced via mutagenesis strategy , and then UGI-T2A-mCherry cassette was inserted through BamHI-EcoRI double digestion strategy. SgRNA expression vectors were U6-sgRNA-EF1alpha-UGI-T2A-mCherry as described previously, and to generate plasmid expressing sgRNAs for puromycin-based enrichment, mCherry was replaced with a puromycin resistance gene. Information for sgRNAs used in this study was listed in Supplementary Data 2 incubator . Twenty-four hours before transfection, HEK293T cells were passaged and plated into six-well or 48-well plates (Corning). Base editor- and sgRNA-expressing plasmids (mole ratio 2:1) were mixed with 2.5 μl (48-well) or 8 μl (six-well) Lipo293TM  for transient transfection. For base editing analysis, cells were plated and transfected in 48-well plates and cultured for another 72 h. Then Flow cytometry  was performed to collect double-positive cells. For transcriptome analysis, cells were plated and transfected in six-well plates. Twenty-four hours later, the medium were replaced with fresh DMEM containing 2 μg/ml puromycin  and cultured for another 48 h, then total RNA were collected for high-throughput RNA sequencing.Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS  were used for HEK293T cells  and cells were cultured at 37 °C in 5% CODouble-positive cells collected as described above were treated with DirectPCR reagent . Targeted amplifications were produced using LA Taq . Sanger sequencing results were quantified with EditR software and targeted amplicon sequencing results were analyzed with CRISPResso2 . Targeted amplicon sequencing was performed using the Illumina NovaSeq platform at Shanghai Personalbio Technology. Experiments for amplicon sequencing were performed in biological triplicates.32, with the addition of several TadAs having reported structures in PDB , Staphylococcus aureus TadA (Q99W51), Salmonella choleraesuis TadA (Q57LE3) and Streptococcus pyogenes TadA (Q5XE14)); (3) evolution distances, 50 TadA orthologs with large evolution distances were included to ensure the high diversity. To design TadA orthologs for functional screening, conserved residues corresponding to A106 and D108 residues of ecTadA were substituted with valine (V) and asparagine (N), respectively, as A106V and D108N are core mutations for ecTadA to be adapted for functional ABE. Overall, six TadA orthologs already have N substitution at the residue corresponding to D108 and only A-V mutation was induced at the residue corresponding to A106 of ecTadA, while one TadA ortholog already have V substitution at the residue corresponding to D108 and only D-N mutation was induced at the residue corresponding to D108 of ecTadA. Additionally, four TadA orthologs have no obvious conserved residues and thus, no mutations were induced. Selected TadA orthologs were referred to as UniProt ID with mutations corresponding to A106 and D108, such as V, N, or VN.To select representative TadA orthologs, TadA orthologs were initially aligned with BLOSUM62 using Jalview2.11.2.5. Selection criteria include (1) >140 a.a. length to exclude potential partial sequences (16 orthologs were excluded); (2) structural information . After removing duplicates with Picard , reads were split at junctions into exon segments and reassigned the mapping qualities from STAR. Base-quality score recalibration (BQSR) was performed as the DNA-seq to remove potential experimental biases. MuTect2 (202010.02) was then used to identify variants, and the VariantFiltration tool (gatk-4.1.4.0) with parameter --filter-expression “QUAL <25|| MQ <20.0|| QD <2.0|| FS >30.0|| DP <20” was used to filter variants with base-quality score <25, mapping quality score <20, Fisher strand values >30.0, qual by depth values <2.0 or sequencing depth <20. Variants in GFP-only expressing cells were considered as SNPs and filtered out from other groups.Total RNA was extracted using Trizol reagent  for RNA sequencing. Illumina Hiseq (PE 2◊150), at a depth of ~20 million reads per sample was used. The long-RNA-seq-pipeline of ENCODE Consortium was used for RNA sequencing analysis, with GRCh38 reference genome using annotation GRCh38.v96 via STAR (v2.4.2a) in 2-pass mode used for alignmentFor H2AX staining, cells were collected and washed with PBS, and fixed with 4% paraformaldehyde at room temperature (25 °C) for 15 min. Following washes with PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 2 min. Next, block cells with buffer (10% goat serum in TBS) at room temperature (25 °C) for 1 h. For the detection of DNA damage, stain the cells with γ-H2AX antibody  at 4 °C for 2 h and wash with PBS. Cells were gated on fluorescein isothiocyanate and allophycocyanin using the Fortessa Flow Cytometer (BD Biosciences), and results were analyzed by FlowJo v10.t-test was used for two group comparisons in Figs. P < 0.05 was considered as statistical significance, with * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. RNA-seq analysis was conducted using STAR (v2.4.2a), Sentieon® genomics tools (v202010.02), Picard (v2.23.6), MuTect2 (202010.02), and VariantFiltration tool (gatk-4.1.4.0). N = 2 to 4 independent biological replicates were performed and demonstrated in each figure. In this study, no statistical method was used to predetermine sample size, and no data were excluded from the analyses. The experiments were not randomized and the Investigators were not blinded to allocation during experiments and outcome assessment.Statistical analyses were performed using Prism 8.4.0 (GraphPad). The results are presented as mean (heatmap) or mean ± standard error of the mean (SEM) (bar plot). Two-tailed unpaired Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


Deinococcus radiodurans, an extremophilic bacterium with extraordinary resistance to oxidative stresses. We identified four proteins that preferably bind to oxidized RNA: PNPase (DR_2063), DEAD box RNA helicase (DR_0335/RhlB), ribosomal protein S1 (DR_1983/RpsA), and transcriptional termination factor (DR_1338/Rho). Among these proteins, PNPase and RhlB exhibit high-affinity binding to 8-oxoG-modified RNA in a dose-independent manner. Deletions of PNPase and RhlB caused increased sensitivity of D. radiodurans to oxidative stress. We further showed that PNPase and RhlB specifically reduce the cellular availability of 8-oxoG-modified RNA but have no effect on oxidized DNA. Importantly, PNPase directly interacts with RhlB in D. radiodurans; however, no additional phenotypic effect was observed for the double deletion of pnp and rhlB compared to the single deletions. Overall, our findings suggest the roles of PNPase and RhlB in targeting 8-oxoG-modified RNAs and thereby constitute an important component of D. radiodurans resistance to oxidative stress.8-Oxo-7,8-dihydroguanine (8-oxoG) is a major RNA modification caused by oxidative stresses and has been implicated in carcinogenesis, neurodegeneration, and aging. Several RNA-binding proteins have been shown to have a binding preference for 8-oxoG-modified RNA in eukaryotes and protect cells from oxidative stress. To date, polynucleotide phosphorylase (PNPase) is one of the most well-characterized proteins in bacteria that recognize 8-oxoG-modified RNA, but how PNPase cooperates with other proteins to process oxidized RNA is still unclear. Here, we use RNA affinity chromatography and mass spectrometry to search for proteins that preferably bind 8-oxoG-modified RNA in IMPORTANCE Oxidative RNA damage can be caused by oxidative stress, such as hydrogen peroxide, ionizing radiation, and antibiotic treatment. 8-oxo-7,8-dihydroguanine (8-oxoG), a major type of oxidized RNA, is highly mutagenic and participates in a variety of disease occurrences and development. Although several proteins have been identified to recognize 8-oxoG-modified RNA, the knowledge of how RNA oxidative damage is controlled largely remains unclear, especially in nonmodel organisms. In this study, we identified four RNA binding proteins that show higher binding affinity to 8-oxoG-modified RNA compared to unmodified RNA in the extremophilic bacterium Deinococcus radiodurans, which can endure high levels of oxidative stress. Two of the proteins, polynucleotide phosphorylase (PNPase) and DEAD-box RNA helicase (RhlB), interact with each other and reduce the cellular availability of 8-oxoG-modified RNA under oxidative stress. As such, this work contributes to our understanding of how RNA oxidation is influenced by RNA binding proteins in bacteria. Reactive oxygen species (ROS) are generated either through basal cellular metabolism or from external environmental exposures to oxidative stresses, such as ionizing radiation, UV radiation, air pollution, and certain chemicals , 2. Impo5–Escherichia coli and its mammalian homologs (such as MutT homolog 1 [MTH1]) cleave ribonucleotides carrying 8-oxoG by hydrolyzing the oxidized nucleoside diphosphates and/or triphosphates to monophosphates  is one of the most prevalent nucleobase oxidations , 11. 8-o12–15–osphates , 19, preosphates . Unlike osphates . Importaosphates , 22, 23.osphates , whereasosphates , 26.E. coli PNPase and human PNPase have been shown to bind 8-oxoG-modified RNA with higher affinity than to unmodified RNA , a conserved 3′-5′ exoribonuclease, is the only characterized RBP that interacts with 8-oxoG-modified RNA in bacteria . Both E.fied RNA , 29. Thefied RNA , 30, 31.fied RNA –34. In Elase Eno . The intlase Eno . For insy PNPase , 36. PNPy PNPase . HoweverDeinococcus radiodurans is a unique bacterium that possesses remarkable tolerance to extreme environmental conditions that generate oxidative damage to macromolecules  and UV radiation  (D. radiodurans is still unclear.olecules . The suradiation . Howeveradiation . InteresDR_1624) . HoweverD. radiodurans exhibit high-affinity binding to 8-oxoG modified RNA relative to unmodified RNA in a dose-independent manner. The deletion of PNPase and RhlB significantly impairs the survival of D. radiodurans under H2O2 stress and results in elevated levels of 8-oxoG-modified RNA (not 8-oxoG-modified DNA). Most importantly, PNPase interacts with RhlB, and no additive effect on survival and 8-oxoG-RNA level was observed in the double-deletion strain of PNPase and RhlB relative to the single-deletion strains. The latter result suggests that PNPase and RhlB may function cooperatively to defend against oxidative stress in D. radiodurans. To the best of our knowledge, this is the first study that indicates the essential role of RBPs in response to cellular damage induced by oxidative stresses in D. radiodurans.Here, through a combination of RNA affinity chromatography, mass spectrometry and biochemical analysis, we demonstrate that PNPase and RhlB in D. radiodurans, we conducted an RNA affinity chromatography approach followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis .To identify RBPs that preferably bind to 8-oxoG-modified RNA in analysis . Two typ2 fold change ≥ 0.58496, P ≤ 0.05) in the pulldown from the 8-oxoG-modified oligonucleotide relative to the control oligonucleotide (D. radiodurans), DR_0335/RhlB (DEAD box RNA helicase), DR_1983/RpsA , DR_1338/Rho (Rho transcription termination factor), DR_0353/Rnr (RNase R), DR_1797/NusA (transcription elongation factor NusA), DR_1836/Ffh , and other three ribosomal proteins . These results suggest that these proteins could possibly directly bind to 8-oxoG-modified RNA. Interestingly, DR_0550/MutT was also found to be enriched in the 8-oxoG pulldown (see Table S1). Considering that DR_0550/MutT does not contain any known RNA-binding domain and can only recognize the oxidized RNA precursor nucleotides, not the RNA molecules oxidized after transcription . Each purified protein was incubated with oligoribonucleotides carrying one 8-oxoG or no 8-oxoG; these mixtures were subjected to RNA affinity chromatography, and pulldown products were subjected to Western blotting. As shown in 43–dK) showed that PNPase and RhlB bind to 8-oxoG-modified oligoribonucleotide with high affinity at the nanomolar range  . In these assays, the same RNA oligoribonucleotides used in the affinity chromatography assay were radiolabeled and incubated with purified PNPase, RhlB, RpsA, and Rho at different concentrations, followed by electrophoresis in native PAGE gels. As shown in Fig. S3 in the supplemental material, all four proteins exhibited higher binding affinity to the 8-oxoG-containing oligoribonucleotide than the control oligoribonucleotide. Importantly, quantification of the dissociation constant (ctively) . In contctively) ; Table 1Previous studies indicate that certain RBPs may have the ability to differentiate mildly from heavily oxidized RNA in HeLa cells , 26. To D. radiodurans, we attempted to completely abolish the expression of these proteins in D. radiodurans using a previously described homologous recombination method , which is contrary to the function of catalase (encoded by katA), the main antioxidant protein in D. radiodurans that removes H2O2 from the cells efficiently under oxidative stress . These results suggest that the reduced cell viability of the Δpnp and ΔrhlB strains is not due to deficient bacterial growth or reduced H2O2 in the cell culture.Importantly, significantly lower levels of PNPase and RhlB expression were detected at both mRNA and protein levels under H2 stress  to D. Itin vivo by PNPase or RhlB deletion. The WT, Δpnp, and ΔrhlB strains were challenged with 0 and 100 mM H2O2 for 30 min, followed by RNA extraction and enzyme-linked immunosorbent assays (ELISAs) using a specific 8-oxoG antibody. As shown in 2O2 exposure relative to no treatment in all strains. Notably, the levels of 8-oxoG-modified RNA detected in all three strains in the absence of H2O2 exposure were comparable. However, increased levels of 8-oxoG-modified RNA were detected in the Δpnp and ΔrhlB strains under H2O2 compared to the WT strain .Given that DNA can also be severely damaged under oxidative stress , 10, we reatment . These rE. coli, either forming an individual complex or as part of the degradosome  experiments, followed by LC-MS/MS analysis, and 584 and 552 proteins were identified in PNPase- and RhlB-coIP experiments, respectively. Among these proteins, 66 proteins were significantly enriched  in the PNPase-coIP compared to the control strain  in the RhlB-coIP , DNA-directed RNA polymerase subunits , as well as many ribosomal proteins (see Table S2). Gene ontology and enrichment analysis revealed that these proteins are involved in diverse pathways, especially ribosome assembly/biogenesis, transcription, and translation processes  are potentially interacting with each other . Importantly, interactions between 8-oxoG recognizing RBPs were also observed: we identified RhlB and RpsA in the coIP of PNPase  was tagged with 3×FLAG at the C terminus on the chromosome to allow detection by Western blotting with an anti-FLAG antibody. As shown in 6-tagged PNPase. Notably, these copurifications were not abolished when cell lysates were treated with RNases, suggesting that the interaction between PNPase with RhlB or RpsA does not rely on their RNA substrates  was constructed, and the response of this strain to the H2O2 stress was evaluated. We found that the ΔpnpΔrhlB strain exhibited similar survival compared to single-deletion strains (Δpnp and ΔrhlB) under H2O2 stress, which is significantly lower than WT under 100 and 200 mM H2O2  and double-stranded RNA (dsRNA) by PNPase in vitro. We used a 22-mer ssRNA and an artificial dsRNA as the substrates in PNPase degradation assays in the presence or absence of RhlB. As depicted in D. radiodurans. The partial degradation of the dsRNA when only PNPase was added  that can preferably bind and affect the cellular availability of the 8-oxoG-modified RNA to protect cells under oxidative stress; this represents another mechanism underlying the extreme resistance of D. radiodurans to oxidative stresses.In the past, extensive studies have been conducted to understand the mechanism of the resistance of ulations , 50–52. iodurans . HoweverE. coli and HeLa cells).8-oxoG modification in RNA can cause a decrease in the fidelity of gene expression or translation and is closely related to a range of diseases , 14, 20.D. radiodurans. Given that only PNPase was previously shown to exhibit preferential binding to 8-oxoG-modified RNA   that selectively bind 8-oxoG-modified RNA in the nonmodel bacterium fied RNA , 29, the and Rho , implyineotides) . It is pD. radiodurans. Indeed, deletion of PNPase and RhlB resulted in reduced survivability of D. radiodurans and elevated 8-oxoG-RNA levels upon H2O2 exposure (pnp and ΔrhlB mutant strains under H2O2 may only be related to the accumulation of oxidized RNA in the cells (not oxidized DNA). Notably, the expression of PNPase and RhlB were observed to significantly decrease at both mRNA and protein levels under H2O2 stress  occurring internally or at the 3′ end of the RNA substrates . Combinit of RNA . Furthere stress  and 5, ae stress  and B. Iin vitro . Based oormation , 49. Morormation , 29, in D. radiodurans, including DNA repair proteins, and RNA polymerase subunits  was also found to interact with PNPase in D. radiodurans in an RNA-independent fashion  can also be copurified with PNPase in D. radiodurans  and its derivatives were cultured aerobically in TGY media  at 32°C or on agar plates when required. E. coli DH10b and BL21(DE3) strains were grown aerobically in Luria-Bertani (LB) media  or plates at 37°C. All strains used in this study are listed in Table S3. When necessary, antibiotics were used at the following concentrations: ampicillin at 100 μg/mL for E. coli, chloramphenicol at 15 μg/mL for E. coli and 3.4 μg/mL for D. radiodurans, and kanamycin at 50 μg/mL for E. coli and 16 μg/mL for D. radiodurans.The D. radiodurans mutants were constructed by double crossover recombination of a kanamycin resistance cassette into the genome to replace the intended mutation region as described previously  strains. The colonies were then confirmed by PCR screening and Sanger sequencing. All plasmids and primers used in this study are listed in Table S3 and S4, respectively.The eviously . Gene ta plasmid . The revGNNNNNGNNNNN8-oxoGNNNNNGNN-3′; N stands for a random base [A/T/C/G]) was chemically synthesized for the RNA affinity chromatography assay, and an unmodified oligonucleotide  was used as a control. To determine whether the binding of PNPase and RhlB is dose dependent, three more RNA oligonucleotides containing different numbers of 8-oxoG were used: 8-oxoG ×2 (5′-biotin-NNNGNNNNNGNNNNN8-oxoGNNNNN8-oxoGNN-3′), 8-oxoG ×3 (5′-biotin-NNNGNNNNN8oxoGNNNNN8-oxoGNNNNN8-oxoGNN-3′), and 8-oxoG×4 (5′-biotin-NNN8oxoGNNNNN8oxoGNNNNN8-oxoGNNNNN8-oxoGNN-3′). Next, 100 mL of D. radiodurans cells  was harvested at an optical density at 600 nm (OD600) of 0.8, washed, and resuspended in lysis buffer (1 mM Tris-HCl [pH 8.0] containing 1 mM phenylmethylsulfonyl fluoride [PMSF]). The cells were then lysed using sonication as described before . Then, 500 μg of the lysates or purified His6-tagged proteins were precleared with 500 μg of Dynabead MyOne Streptavidin C1 (Thermo Fisher Scientific) in 300 μL of binding buffer  for 30 min at 4°C with rotation in the presence of 0.4 U/mL SUPERase-In RNase Inhibitor (Thermo Fisher Scientific) and 125 μg of yeast tRNA (Sigma-Aldrich). Then, 10 μg of biotinylated modified/unmodified RNA baits in 20 mM Tris-Cl (pH 7.5) was refolded by heating at 90°C for 10 min, followed by incubation with precleared lysates or His6-tagged protein (200 μg) for 30 min at room temperature and then for 2 h at 4°C. The mixture was then added to 500 μg of Dynabeads MyOne Streptavidin C1 preblocked with BSA (1%) and tRNA (50 μg/mL) for 2 h at 4°C. The RNA-protein complexes were pulled down and washed with 900 μL of wash buffer  three times. The bead pellets were then eluted using 20 μL of high-salt elution buffer . To increase the yield, the elution step was repeated, and the eluates were combined. The eluted proteins were then analyzed by SDS-PAGE, Western blotting, or LC-MS/MS.To identify specific 8-oxoG binding proteins, a 24-mer RNA oligonucleotide  as described previously  by T4 polynucleotide kinase (New England BioLabs) and purified using an Oligo Clean & Concentrator kit (Zymo Research). Next, 10 fmol of radiolabeled RNA probes  was incubated with increasing amounts of each purified proteins  in 10 μL reactions in 1× binding buffer . When testing PNPase, final protein concentrations of approximately 0 to 1,000 nM were used, and the incubation was performed in TMK buffer . Reaction mixtures were incubated at 37°C for 1 h and then resolved by native electrophoresis in 0.5× Tris-borate-EDTA running buffer at 4°C for 3 h at 120 V using 5% polyacrylamide native gels. The gels were dried onto 3MM Whatman paper, exposed to a phosphor screen, and visualized using a Typhoon FLA 700 . The signals were analyzed, and the equilibrium dissociation constant (dK) was determined as described earlier  were grown to an OD600 of 0.8 and treated with formaldehyde (1% [wt/vol]) at room temperature for 15 min with continuous shaking to facilitate the cross-linking. Glycine was then added to a final concentration of 0.125 M to quench the cross-linking reaction. Cross-linked cells (50 mL) were washed three times with ice-cold binding buffer . Cells were then resuspended and lysed by sonication as described above. After lysis, the crude extracts were centrifuged at 12,000 rpm for 10 min at 4°C to collect the supernatant. For each His6-tag pulldown reaction, 1 mL of cell extracts containing 500 μg of protein was incubated with 50 μL of Ni-NTA magnetic beads (New England BioLabs) for 30 min at room temperature. The protein-protein mixtures were then washed using 500 μL of wash buffer  three times and eluted using 20 μL of elution buffer . To increase the yield, the elution step was repeated, and the eluates were combined. The protein samples were then concentrated by dialysis and subjected to LC-MS/MS or Western blotting. To determine whether the protein interactions are RNA-dependent, one set of samples was incubated with 2 μg of RNase A and 5 U of RNase T1 (Thermo Fisher Scientific) for 15 min at 22°C after cell lysis. coIP samples from cell lysates treated with or without RNase were subjected to Western blotting as described below.pRADgro plasmids carrying HisD. radiodurans R1 (ATCC 13939) database using Sequest HT in Proteome Discoverer 1.4 and normalized to the total ion intensity. The identifications were validated with Scaffold v4.4.1 (Proteome Software) with >99.0% probability and with a minimum of two peptides at 99.0% peptide probability. The false discovery rate was set at 1% for peptides and 5% for protein identification. The difference between two strains or fractions was calculated by using a Student t test, and the P values were adjusted by the Benjamini-Hochberg method . The mixtures were denatured at 95°C for 5 min before being run ~3 mm into the 12% Mini-Protean TGX precast protein gels (Bio-Rad). After staining with Coomassie Bright Blue (Sigma-Aldrich), the protein bands were excised using a clean razor blade. The gel tubes were then dehydrated with 100% acetonitrile (Sigma-Aldrich), reduced with 10 mM dithiothreitol (DTT) for 30 min at room temperature, followed by alkylation with 50 mM iodoacetamide in the dark for another 30 min. The gels were washed, rehydrated, and then digested with 10 ng/μL trypsin (Thermo Fisher Scientific) overnight at 37°C. Proteins were then extracted from the gels using 5% formic acid and 1:2 (vol/vol) 5% formic acid-acetonitrile. The samples were sent for LC-MS/MS analysis at the University of Texas ICMB proteomics facility. The resulting protein spectral counts were searched against the UniProt g method .6 tag monoclonal antibody  at room temperature. Afterward, the membranes were further incubated with anti-mouse IgG(H+L)/HRP conjugate (Promega) for another 1 h. After three washes with TBST, chemiluminescence was detected by using Clarity Western enhanced chemiluminescence substrate (Bio-Rad). Protein samples were visualized by Coomassie staining in parallel to show the equivalent protein loading in each lane. Quantitation of Western blot band intensities was done using the CLIQS software . Each Western blot analysis was performed at least three times.Protein samples were resuspended in the SDS sample buffer, boiled at 95°C for 10 min, separated by 12% Mini-Protean TGX precast protein gels (Bio-Rad), and transferred to nitrocellulose membranes (Bio-Rad) using a Trans-Blot semidry transfer cell (Bio-Rad). Membranes were blocked with 5% milk powder overnight at 4°C and incubated for 1 h with monoclonal Anti-Flag M2-peroxidase antibody  or HisD. radiodurans strains  were grown to an OD600 of 0.8 and stressed with different dosages (0 to 200 mM) of H2O2 (Sigma-Aldrich) for 30 min at 4°C in the dark. The cells were serially diluted (10−3 to 10−5) with sterilized 1× phosphate-buffered saline and plated on TGY agar plates with appropriate antibiotics. The plates were incubated at 32°C for 3 days, and the colonies were counted. The relative survival was calculated as the percentage of the number of colonies under each H2O2 concentration compared to that for no H2O2 treatment.D. radiodurans strains  were grown to an OD600 of 0.8, stressed by 100 mM H2O2 (Sigma-Aldrich) for 30 min at 4°C in the dark, and then collected by centrifugation at 12,000 × g for 5 min. Total RNA was extracted and purified using the Direct-zol RNA miniprep kit (Zymo Research) according to the manufacturer’s protocol. DNA contamination in the RNA samples was removed by DNase digestion during the extraction. Total DNA was extracted using a Wizard genomic DNA purification kit (Promega), including an RNase digestion step to remove the RNA contamination in the samples. The levels of 8-oxoG in DNA and RNA samples were measured by a competitive ELISA  as described previously  were grown to an OD600 of 0.8 and stressed by 0 or 100 mM H2O2 (Sigma-Aldrich) for 30 min at 4°C in the dark. The H2O2 level in the cultures was immediately determined using an Amplex Red kit (Thermo Fisher Scientific) as recommended by the manufacturer.D. radioduransstrains were evaluated using a Plate Reader (BioTek). Biological triplicates of each strain were distributed into 96-well plates with 200 μL TGY media. The initial OD600 was adjusted to 0.1, and the turbidity (600 nm) was measured every 30 min for 8 h as the cultures grew with shaking at 32°C. Catalase (Sigma-Aldrich) was added to a final concentration of 50 or 100 nM when needed.Growth curves of in vitro was performed as described previously (GGUACUGCGCUCUACAGUAUUU-3′) was radiolabeled at the 5′ end using 10 μCi of [γ-32P]ATP (Perkin-Elmer) and T4 polynucleotide kinase (New England BioLabs). To generate dsRNA, the labeled 22-mer RNA oligonucleotide was incubated with an unlabeled 11-mer RNA oligonucleotide  in RNA annealing buffer  by heating to 95°C for 5 min, followed by slow cooling (≫1 h) to room temperature. To maximize the hybridization efficiency, the 22-mer RNA oligonucleotide and the 11-mer RNA oligonucleotide were incubated at a ratio of 1:1.5 (mol/mol). 10 nM  of double-stranded RNA duplex or 22-mer single-stranded RNA was incubated with PNPase  or RhlB  alone or with mixtures of PNPase and RhlB at 32°C for 30 min in 10 μL of activity buffer . The reaction was then stopped by adding 10 μL of 2× RNA loading buffer (New England BioLabs). The samples were separated on a 20% polyacrylamide native gel and visualized using Typhoon FLA 700 .The combinational effect of PNPase and RhlB on structured RNA eviously . A 22-me–ΔΔCT method  on a ViiA7 instrument (Applied Biosciences). cDNA was synthesized using 50 ng of RNA at 55°C for 10 min, followed by denaturation at 95°C for 1 min and 40 cycles of 95°C for 10 s and 60°C for 30 s. 16S rRNA was used as the internal normalization control. The relative expression of each gene was calculated using the 2T method . The expP ≤  0.05, 0.01, or 0.001 were considered to be statistically significant (*), highly significant (**), or extremely significant (***), respectively, as indicated by asterisks in the figures.Student two-sided t-tests were used to assess the significance of the results. Values of PXD028861 (RNA affinity chromatography) and PXD028866 (PNPase- and RhlB-coIP).The LC-MS/MS raw data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository  under th


C. elegans, a family of Vasa-related Germ Line Helicase (GLH) proteins localize in and promote the formation of nuage. Previous studies have implicated GLH proteins in inherited silencing, but direct roles in small-RNA production, Argonaute binding, or mRNA targeting have not been identified. Here we show that GLH proteins compete with each other to control Argonaute pathway specificity, bind directly to Argonaute target mRNAs, and promote the amplification of small RNAs required for transgenerational inheritance. We show that the ATPase cycle of GLH-1 regulates direct binding to the Argonaute WAGO-1, which engages amplified small RNAs. Our findings support a dynamic and direct role for GLH proteins in inherited silencing beyond their role as structural components of nuage.Germline Argonautes direct transcriptome surveillance within perinuclear membraneless organelles called nuage. In   Dai et al. show that worm VASA homologs, GLH proteins, long known as factors that promote perinuclear nuage formation in germ cells, are in fact direct participants in Argonaute surveillance. GLH proteins directly bind mRNAs targeted by Argonautes and utilize their ATPase domains to regulate both mRNA and Argonaute binding. C. elegans anti-sense small RNAs (22G-RNAs) produced by cellular RNA-dependent RNA polymerases (RdRPs) engage WAGO and CSR-1 Argonautes to target nearly all germline-expressed mRNAs. Both RdRP templating and Argonaute surveillance are thought to occur within perinuclear liquid-like condensates called nuage or P granules  through the canonical RNAi pathway, which employs a distinct upstream Argonaute, RDE-1 . WAGO 22C. elegans, the DEAD-box protein RDE-12 functions with the Argonautes RDE-1 and WAGO-1 to promote 22G-RNA production during RNAi  family proteins in the regulation of germline RNAs . GLH-1 hhenotype . glh-4-neratures . Mutationg PGL-1 . Several mutants . GLH pro mutants . In addi mutants .in vitro but does not prevent GLH-1 RNA binding as measured by both in vivo CLIP and in vitro gel-shift assays. Rather, GLH-1(K391A) binding is enhanced on WAGO target RNAs in vivo. A GLH-1 DQAD lesion, predicted to prevent ATP hydrolysis, causes GLH-1 to bind RNA more strongly in vitro. However, interestingly, GLH-1(DQAD) loses its preference for WAGO target binding in vivo and instead binds many abundant mRNAs including CSR-1 targets.Here, we show that GLH-1 and GLH-4 interact with the Argonautes PRG-1 and WAGO-1, as previously reported . We showglh-1 cause a loss of 22G-RNAs on WAGO targets and ectopic 22G-RNAs on many other target mRNAs. The ectopic 22G-RNAs depend on GLH paralogs and PRG-1 activity, suggesting that GLH-1 prevents other GLH paralogs from inducing piRNA-initiated overproduction of 22G-RNA on many targets. Third, we show that GLH-1(K391A) strongly impairs transgenerational silencing and WAGO small-RNA production without disrupting P granules or other Argonaute small-RNA levels. GLH-1(K391A) exhibits a marked increase in its association with the WAGO-1 protein both in vitro and in vivo, suggesting that when unable to bind ATP, GLH-1 prevents WAGO-1 or co-factors from engaging GLH paralogs, but does so without disrupting GLH protein scaffolding functions required for other Argonaute pathways in nuage. Taken together, our work supports a model in which GLH proteins are recruited by Argonautes to bind and retain target mRNAs within nuage-promoting pathway-specific transgenerational inheritance of small-RNA signals.Our findings suggest that, along with its paralogs, GLH-1 promotes and regulates Argonaute-mediated mRNA surveillance in at least three ways. First, special alleles and double mutants that disrupt P granules also disrupt small-RNA levels in the three major nuage-associated Argonaute pathways—i.e., WAGO, CSR-1, and PRG-1—suggesting that GLH proteins help establish a scaffold for Argonaute-mediated surveillance. Second, null alleles of prg-1 and wago-1 genes. We then used FLAG-specific antibodies to recover co-precipitated factors followed either by liquid chromatography-tandem mass spectrometry (LC-MS/MS), or by denaturing or native polyacrylamide gel electrophoresis (PAGE) to separate bands containing prominent co-factors for immunoprecipitation mass spectrometry (IP-MS)  . Using t (IP-MS) . A simil (IP-MS) .cdk-1::gfp reporter, whose silencing depends on an artificial anti-gfp piRNA , in-frame deletion of the zinc-finger domains from GLH-1 (∆ZF), or specific amino acid substitutions in conserved residues of GLH-1 predicted to prevent ATP binding (K391A) or ATP hydrolysis  (Previous studies found that GLH-1 and GLH-4 function together . To inveto DQAD) . Mutatioto DQAD) , whereasto DQAD) .glh-1 did not initially desilence the piRNA sensor or the WAGO-pathway reporter, but both reporters were expressed in a fraction of glh-1-null worms by the fourth homozygous generation, and the percentage of expressing animals increased in subsequent generations  worms had no effect on silencing  mutants. These results show that GLH-1 and GLH-4 function redundantly in small-RNA silencing.The fact that GLH-1 K391A and DQAD point mutants show more severe phenotypes than null mutants suggests that the mutant K391A and DQAD proteins might interfere with the function of paralogs—e.g., GLH-4—which could otherwise partially compensate for the loss of GLH-1 . Indeed,neration , a desilglh-1-null mutants have an intact RNAi response in animals exposed to dsRNA, but exhibit reduced transmission of silencing to unexposed progeny, an RNAi inheritance defect , glh-1-null animals gradually recovered  defect (glh-1(K391A) worms did not show a csRNAi defect, but instead remained 100% sensitive to RNAi at all temperatures , but glh-1(K391A) did not, we wondered whether misdirection of the PRG-1 pathway might cause the csRNAi defects. Consistent with this idea, a prg-1 loss-of-function mutation completely suppressed the csRNAi defects of glh-1 and glh-4-null mutants (We were surprised to find that presumptive null (deletion or degron) alleles of ) defect –S1F. Noteratures . Since g mutants  and S1F. mutants .glh mutations with their effects on GLH protein localization and P-granule integrity, we inserted GFP coding sequences into the endogenous glh-1 and glh-4 alleles and analyzed their expression. As expected, both GFP:GLH-1 and GFP:GLH-4 localized to perinuclear P granules  forms aggregates, we controlled the expression of GFP:GLH-1(DQAD) by introducing an in-frame, auxin-inducible degron . We mainglh mutant worms. Although GLH-1 has been reported to interact with DCR-1  mapping to each gene in the input and IP datasets. A gene was scored as enriched if the RPM level in the IP increased by 2-fold over the level in the corresponding input dataset. In wild-type animals, approximately equal numbers of germline genes were enriched in the WAGO-1 and CSR-1 IPs  did not exhibit ectopic 22G-RNAs on CSR-1 targets  were among the 1,825 genes depleted 2-fold or more of WAGO 22G-RNAs in glh-1(K391A), further supporting the idea that GLH-1(K391A) blocks the ability of GLH paralogs to promote WAGO 22G-RNA biogenesis (Deleting two or more ogenesis F.IP-western blot assays confirmed that GLH-1 and GLH-4 co-precipitate with PRG-1 and WAGO-1  and 5B. To ask whether the stronger-than-null phenotypes of GLH-1(K391A) reflect changes in its protein interactions, we used mass spectrometry to identify proteins that co-precipitate with wild-type GLH-1 or with GLH-1(K391A). This analysis revealed that the K391A lesion does not significantly perturb GLH-1 interactions with other GLH proteins. We were intrigued to note, however, that PRG-1 and WAGO-1 were enriched in GLH-1(K391A) immunoprecipitates . We confin vitro binding assays between WAGO-1 and GLH-1 proteins. We used MBP:GFP as a negative control for specificity. Lysates containing MBP:WAGO-1 or MBP:GFP were mixed with lysates containing wild-type or mutant GLH-1 protein and incubated with or without ATP at 20°C for 20 min. In MBP pull-downs, MBP:WAGO-1 co-precipitated more GLH-1(K391A) than wild-type GLH-1 (The above findings suggest that ATP binding by GLH-1 regulates a direct (RNA-independent) interaction with WAGO-1. To explore this possibility, we tagged WAGO-1 with the maltose-binding protein (MBP) and performed pe GLH-1 , consistpe GLH-1 . The conpe GLH-1 . These din vitro. To check for RNA binding, we incubated wild-type or mutant GLH-1 proteins with fluorescently labeled 22-nt single-stranded RNA (ssRNA) oligo and visualized resulting protein/RNA complexes as gel shifts by native PAGE. We found that both GLH-1 wild type and K391A showed weak binding to the ssRNA oligo, whereas GLH-1(DQAD) showed strong ATP-dependent binding  worms were grown to adulthood in the presence of auxin to deplete the toxic protein, after which auxin was removed to allow GLH-1(DQAD) to accumulate in P granules for either 2 h or 4 h before crosslinking.To determine whether GLH proteins bind RNA IP assay  to analyUsing a 2-fold enrichment over background binding, CLIP-sequencing analyses identified 3,120 GLH-1(WT), 3,927 GLH-1(K391A), 4,439 GLH-1(DQAD), and 6,158 GLH-4 targets . The majTo explore the relationship between GLH–mRNA binding and 22G-RNA levels, we analyzed the subset of mRNAs whose 22G-RNA levels decreased by at least 2-fold in GLH-1(K391A) mutants. We chose this group of targets because the K391A mutant strongly depletes a subset of PRG-1-induced WAGO 22G-RNAs that depend on GLH-1 and/or its paralogs  without eliminating scaffolding functions required for other P-granule-associated Argonaute pathways. We used the remainder of germline mRNAs as a control set. To reveal how CLIP binding distributes along the length of mRNAs, we performed a meta-analysis of GLH binding along GLH-dependent or control mRNAs see . As expeGLH-1(DQAD), in contrast, showed a strikingly different pattern of mRNA binding. At 2 h after auxin removal, when GLH-1(DQAD) was localized in perinuclear nuage , DQAD prglh-1-null mutant , GLH-1(DQAD), exhibited enhanced RNA binding in vitro (as expected) and exhibited Argonaute-non-specific association with the 3′ ends of mRNAs in vivo.A poorly understood aspect of transgenerational silencing is how targeting by a primary Argonaute (PRG-1 for piRNAs and RDE-1 for the dsRNA response) leads to the amplification of heritable secondary small RNAs. Here, we have shown that GLH proteins—members of an expanded family of DEAD-box proteins related to drolysis . Surprisglh-1-null mutants showed a csRNAi defect in the exposed generation, which correlated with overproduction of small RNAs on endogenous genes, this defect was suppressed by removing prg-1 activity. Thus, our findings support a model in which GLH proteins are not general factors required for amplification of the silencing signal but rather function more directly to promote transgenerational silencing. Perhaps upstream Argonautes, including RDE-1, PRG-1, and transgenerationally inherited WAGOs, recruit GLH proteins to bind their mRNA targets within nuage to initiate transgenerational silencing. Initial binding of GLH proteins could serve to retain target mRNA in nuage or to promote its conversion to template RNA required for amplification of transgenerational signals that are transmitted along with nuage to offspring.Our findings are consistent with previous work implicating a role for GLH proteins in the transmission of silencing signals to offspring . GLH-1 reside in the largest subdomain of nuage (the P-granule domain), while many components required for amplification of silencing signals reside in smaller subdomains known as Mutator foci . Our finIn insects and mammals, Piwi Argonautes amplify epigenetic signals on transposon RNAs via an amplification cycle in which the cut site of a loaded Argonaute creates the 5′ end of a guide RNA that is loaded onto a downstream Argonaute. This downstream Argonaute can then cleave a corresponding anti-sense transcript to regenerate the upstream guide, and so on. Interestingly, this ‘‘ping-pong’’ amplification cycle requires the DEAD-box protein VASA, and studies in insect cells have linked VASA ATP binding with formation of a Piwi complex, called the ‘‘amplifier complex’’ . A VASA-glh-1-null mutants, as ectopic piRNA-dependent WAGO silencing could reduce availability of WAGO machinery to function in canonical RNAi induced by exogenous dsRNA. Alternatively, piRNA-dependent mistargeting could silence one or more genes that encode protein effectors of WAGO silencing, as suggested in recent studies by Here we have shown that the VASA homologs, the GLH proteins, which are required for P-granule assembly and homeostasis  appears similar to its wild-type localization, we cannot rule out changes in other nuage factors that associate with it; for example, GLH-1(K391A) might reside in a functionally distinct domain of nuage that looks similar to wild-type P granules. Additionally, the biochemical studies reveal interactions that occur within a subset of GLH complexes that are soluble under the extraction conditions used to prepare lysate. Thus, the IP-MS studies and western blotting assays are likely to miss significant interactions that occur in complexes excluded from the analyses. Finally, assays on bacterially purified proteins will unavoidably lack endogenous co-factors or covalent modifications that regulate RNA binding and unwinding properties of the proteins analyzed.Craig.Mello@umassmed.edu).Further information and requests for resources and materials should be directed to and will be fulfilled by the lead contact, Craig Mello  in lysis buffer . The same experimental procedures were also performed for N2 worms as negative controls. Protein extracts were incubated with 3ul anti-FLAG antibody  and 50ul Protein-G magnetic beads (Thermo Scientific) at 4°C for 2 h. Prior to elution, pull-downed components were pretreated with RNase for removing proteins bridged by RNAs. Immunoprecipitates were released from beads by TEV protease cleavage (Thermo Scientific). Finally, the purified protein complex were resolved by SDS-PAGE and visualized by Silver staining (Pierce). The elutes were precipitated by acetone and air dried. Protein samples were re-suspended and digested with Trypsin. LC-MS/MS were conducted at Mengqiu Dong’s lab in NIBS. MS data were processed as described previously  or dsRNA targeting 16 tested genes for over 10 generations  in each generation. RNAi that causes lethality was indicated in RNAi assays were performed by growing worms on HT115 erations . SynchroC. elegans strains were produced using CRISPR-cas9 RNP methods previously described  in 100 worms were checked for each generation. For cold-sensitive RNAi assay, synchronized animals were fed with pos-1 RNAi or L4440 RNAi and grown at 15°C, 20°C and 25°C respectively until they laid sufficient eggs for scoring.For inheritable RNAi assay, synchronous population of worms were exposed to Synchronized populations were bleached and plated on NGM plates with OP50 at 20°C. Brood sizes and hatching rates were measured by counting eggs laid onto the plates and percent of viable embryos. Progenies of at least 15 animals were scored for each strain.Images of GFP-tagged or mCherry-tagged P granule components were captured by an Axio Imager M2 microscope (Zeiss). Worms were constrained on RITE-ON glass slides (Beckton Dickinson) with 1 mM levamisole. Images were further processed and cropped by ImageJ programs. GFP:AID:GLH-1(DQAD) worms were kept on the plates with 500 mM indole-3-acetic acid  for complete depletion of GLH-1 proteins until they grow to young adults. The animals were then transferred to regular plates and allow de novo expression of GLH-1(DQAD) over the time for imaging.Total RNAs were extracted from synchronous population of worms by Trizol (Sigma Alrich) and isopropanol precipitation. Small-RNAs were further purified by mir-Vana miRNA isolation kit (Thermo Scientific). Small-RNA cloning was carried out as previously described . In brieOverall sequencing data were analyzed using previously described pipeline . Raw seq50,000 synchonized young adult worms were homogenized in lysis buffer  and the lysates were incubated with 2ul FLAG antibody  together with 30ul Protein G beads (Thermo Scientific) for FLAG-tagged WAGO-1 IP, or with GFP nanobody (Thermo Scientific) for GFP-tagged PRG-1 IP. Lysates were per-treated with RNase I (NEB) for 20 min at 20°C. After washing with lysis buffer, immunoprecipitates were eluted with 4X SDS sample buffer (BioRad) and were resolved by SDS-PAGE later transfer to PVDF membranes (BioRad). GLH-1 antibody (1:5000), GLH-4 antibody (1:2000), anti-FLAG HRP-conjugated antibody  were used for probing the blots.E. coli were collected and resuspend them in lysis buffer . Bacterial homogenates were then sonicated and went through MBP-trap affinity column  by AKTA. With stringent washing with lysis buffer, protein complex were eluted with 10 mM Maltose (Sigma Aldrich). Resolve elutes by SDS-PAGE and transfer to PVDF membrane for western bloting. Anti-FLAG HRP antibody  and anti-MBP antibody  were used for probing the blots.MBP:WAGO-1, MBP:GFP, GLH-1(WT) and its variants were constructed onto pET-Duet1 vectors. FLAG-tagged GLH-1(WT), FLAG-tagged GLH-1 variants, MBP-tagged GFP and MBP-tagged WAGO-1 proteins were expressed in BL21 cells following 0.5 mM IPTG treatment for 18 h at 18 C. Full-length FLAG-tagged GLH-1 was cloned into pET28a vector. The point mutations K391A and E500Q were introduced by site-directed mutagenesis (NEB). All constructs were expressed in ER2566 E.coli cells (WEIDI) at OD600 of 0.8 with 0.5 mM IPTG for 18 h at 18°C. Cells were resuspended in lysis buffer  and was sonicated on the ice. Then the lysates were treated with 10 μL Ambion RNase Cocktail (Invitrogen) and 10 μL Micrococcal Nuclease (NEB) for removing bacterial RNAs that remain bound to the proteins. After high-speed centrifugation, the supernatant was loaded onto a column with 1 mL Anti-DYKDDDDK G1 Affinity Resin (GenScript) and the 1×FLAG-tagged proteins were affinity extracted. Then the resin was washed with 60 mL wash buffer  and the proteins were eluted with 2 mL elution buffer . After affinity purification, eluted samples were run on Superdex 200 (Sigma Aldrich) in gel filtration buffer  to separate aggregates with monomers according to their molecular weight difference –S5H. AllRNA binding activities of purified GLH-1 WT, K391A and E500Q mutants were assessed by incubating 10 μM monomeric proteins and 2 μM 5′ end Cyanine-3 (Cy3) labeled RNAs  in the presence and absence of 2 mM ATP for 30 min at RT in binding buffer . The RNA-protein complexes were separated on native 8% polyacrylamide gel at 60 V in 13TBE buffer in cold room. Fluorescence signals were detected by BioRad imager.2, 2 mM ATP) over the time. Unlabeled 11 nt RNA was added to a final concentration of 0.125 mM to prevent re-annealing of unwound products. The reactions were stopped by adding EDTA and the proteins were digested by adding Proteinase K (NEB) at 37°C for 20 min. RNA duplex and unwound products were subjected to native 15% polyacrylamide gel at 90 V in 1×TBE buffer at room termperture. Fluorensent Cy3 signals were detected by a BioRad imager.Helicase unwinding assays were modified from previously described method . RNA dupglh-1 allele and were used to account for unavoidable background RNA binding to the IP matrix. Independent synchronized populations of young adult animals were exposed to UV light and then subjected to extraction, partial RNase digestion (Thermo Scientific), and FLAG protein immunoprecipitation (Sigma Aldrich). Electrophoresis on a denaturing poly-acrylamide gel was then used to separate the proteins in the IP complex, followed by excision of a gel-slice corresponding in size to the GLH protein (and any associated cross-linked RNA fragments). RNA was then eluted from the gel slice and subjected to library construction and high-throughput sequencing.CLIP assays were modified from previously described CLIP methods . Two indglh-1(K391A) worms and the transcripts with unaltered 22G-RNA levels. Distribution of CLIP-seq reads along the transcripts from 5′ to 3′ was plotted using deeptools.3′ adaptors were trimmed from raw data by Cutadapt and the remaining reads with at-least 15nt-long were kept for later analysis. Processed CLIP sequences were mapped to both genome and transcriptome of Wormbase WS262 using STAR genome aligner. Percentage of reads for each RNA class was calculated by dividing sum of mapped reads with total mapped read counts for the same sample. Relative aggregated CLIP reads mapped to each gene were calculated using HTseq and normalized by total read counts. GLH targets were scored by implementing filters for genes with at least two-fold enrichment of CLIP reads over untagged input data and minimal 5 RPM. To visualize CLIP binding over these target mRNA cateogires we generated a composite plot where CLIP reads from all genes in each group were plotted along a 5′ to 3′ metagene space, where each interval on the x axis represents 1% of the total mRNA length. Experimental and control target space were defined by mRNAs with over 2-fold reduction of 22G-RNAs in Sample sizes of each assay were described in figure legends, 1