Drosophila melanogaster, insulin/insulin-like growth factor (IIS) and mTOR-mediated signaling within adipocytes regulates oogenesis. To facilitate comparative study of nutrient-sensing pathway activity in the fat body, we developed antibodies to assess IIS (anti-FOXO) and mTOR signaling (anti-TOR) across three nymphalid species (Lepidoptera). By optimizing whole-mount fat body immunostaining, we find FOXO nuclear enrichment in adult adipocytes, like that observed inDrosophila. Additionally, we show a previously uncharacterized TOR localization pattern in the fat body.Nutritional stress impacts many insect species that have differing reproductive strategies and life histories, yet it is unclear how nutrient-sensing signaling pathways mediate tissue-specific responses to changes in dietary input. In Drosophila melanogaster. For example, nutrient sensing pathways, like IIS, mTOR, and the amino acid response (AAR) pathway, function within adultDrosophilaadipose tissue to control distinct stages of oogenesis . However, insects as a group that potentially includes 5.5 million species  have diverse life histories. For example, female reproductive strategies are influenced by differences in ovarian dynamics , egg carbon:nitrogen ratios , and ovariole numbers . Insects also vary in distribution of the adipose tissue within the body, which could potentially affect inter-organ communication.Drosophila is therefore an excellent model for one such suite of traits but is not representative of all insects. We must develop additional diverse insect models in order to understand the variety of mechanisms controlling life history responses to dietary stress across insects.Resource allocation is inextricably linked to tradeoffs between life-history traits, allowing organisms to adapt to a dynamic environment . Nutritional control of the tradeoff between survival and reproduction is one of the best-characterized examples of this phenomenon  and has been described for a variety of organisms, including insects. In several insect species, nutrients are reallocated from the energy intensive process of oogenesis to support somatic tissue maintenance during nutrient deprivation . This ensures that organisms survive beyond suboptimal nutritional conditions and do not waste resources on reproduction when the likelihood of offspring survival is low. While it is likely that multiple tissues sense and respond to nutrient input, we are just beginning to understand how inter-organ communication mediates whole organism responses in the heavily studied insect model,Drosophila melanogasterand various butterfly species within the Nymphalidae (Lepidoptera). These species differ in fat body distribution , ovarian dynamics , egg C:N ratio  and completeness of the adult diet .Given that nutrient sensing signaling pathways control the response of insect fat body to changes in dietary input, which in turn affects allocation to egg production and other life history functions , we are interested in comparing the operation of these pathways and their outcomes amongDrosophila.Upon insulin receptor activation, Akt, the main effector kinase, acts on several targets to regulate cellular metabolism, survival, proliferation, and growth as well as reproduction . When phosphorylated by Akt, the transcription factor FOXO is restricted from the nucleus, where it controls expression of genes that mediate the negative effects of reduced IIS . In addition, Akt indirectly activates the mTOR kinase by inhibiting the negative regulatory complex of mTOR, Tsc1/Tsc2 . The mTOR kinase can also be activated by the Ragulator complex which senses amino acid status . In both cases, mTOR activation happens at the lysosome.The response to dietary stress of the IIS pathway has been well characterized inDrosophila, the transcriptomes for our non-model lepidopteran species have very limited similarity to those ofDrosophila melanogaster. Therefore, the likelihood of successful use ofD. melanogasterantibodies in the lepidopteran species is low. In contrast, our identified FOXO and TOR transcriptomes for the three lepidopteran species had high percent similarity (> 95%), indicating likely success in developing an antibody for each protein that would work in all three butterfly species.To gain a better understanding of how IIS functions within lepidopteran fat tissue, we want to visualize changes in localization of FOXO and mTOR. One approach to tracking localization of these components is to use species-specific antibodies for immunocytochemistry. While the required antibodies for FOXO and mTOR are available forAgraulis vanillae andHeliconius charithonia, we first determined the protein sequences for those genes for each species. We downloaded amino acid sequences forBombyx mori(Lepidoptera: Bombycidae) andHeliconius melpomene melpomene for FOXO and TOR from uniprot.org. We then used GeneiousPrime to generate transcriptome sequences for these proteins. We blasted these sequences againstA. vanillae andH. charithoniatranscriptomes to locate the sequences for each gene for these species. Transcriptome data forH. charithoniacame from Catalan et al (2018), and forA. vanillaefrom Hanly et al (2019). Additionally, we used transcriptome and amino acid sequences forSpeyeria mormonia, because we are interested in experiments with that species in the future. These RNA sequences were converted to amino acid sequences for determination of epitopes joint among the species. Epitope identification and antibody production were done by Genscript. Rabbits were used for polyclonal antibody production. We also used GeneiousPrime to compare the similarity of FOXO and TOR RNA sequences among our three species, as well as withDrosophila melanogaster(Diptera: Drosophiladae).In this report, we describe the development of Lepidoptera-specific antibodies for FOXO and mTOR to track changes in localization of FOXO and mTOR. To generate antibodies for FOXO and TOR inDrosophilasince adipocytes in fruit flies from nutrient-rich conditions show low-nearly undetectable levels of FOXO nuclear localization . The majority of mTOR immunoreactivity shows diffuse cytoplasmic localization with slight enrichment at cell-cell junctions . Under nutrient replete conditions, mTOR localizes to lysosomes where it can be activated by amino acids . Thus, future studies will assess if the mTOR staining pattern in butterfly adipocytes colocalizes with lysosomal markers. For both FOXO and mTOR immunolabeling, we are confident that the observed staining patterns are specific since there is no fluorescence in samples incubated without primary antibody . Moreover, the nuclear localization of FOXO is quite different from the cytoplasmic localization of mTOR . There is stronger fluorescence intensity at the lowest antibody concentration for tissue treated with Tween 20 than that treated with Triton X-100 . At higher antibody concentrations, detergent type is not a major factor in immunostaining quality. Taken together, we achieve the best signal-to-noise ratio for both antibodies when using Tween 20 as the detergent and 2.5 μg/mL of anti-FOXO and 5 μg/mL of anti-mTOR .We used a standard whole-mount immunostaining protocol to test immunoreactivity of these antibodies in lepidopteran fat body. We varied antibody concentration  and permeabilization solution detergent (Triton X-100 versus Tween 20). We find that both antibodies show immunoreactivity in lepidopteran fat body at all three concentrations with varying robustness  in all three butterfly species tested. For both antibodies, higher concentrations produced stronger signal . FOXO immunoreactivity showed enrichment in the nucleus with diffuse labeling in the cytoplasm . Since FOXO translocation to the nucleus results from reduced IIS pathway activity, this data suggests that the butterflies from which the fat body originated may have been under nutrient stress. Alternatively, lepidopterans may have a higher baseline of FOXO-dependent gene expression thanDrosophilato other insects that differ in their fat body distribution, mode of ovarian function (e.g. stem cell-supported versus not), and adult diet macronutrient composition?In the future, we will further optimize the immunostaining protocol by modifying additional steps in the protocol, such as fixative type and fixation time. This work demonstrates the feasibility of using these newly developed lepidopteran antibodies to answer questions like how generalizable are nutrient sensing mechanisms inButterfly collectionAgraulis vanillae females were collected in Sunrise, Florida in January 2022.Heliconius charithonia females came from Davie, Florida and south Texas in January 2022 and fall 2021 respectively.AntigensFOXO: MSLPRGGSYQSPWSSQTALSELEGAMGEFEPVGELAEVGFEPQTRARSNTWPQPRPENYVDAEDPGSKKNSNQNLSGAPPLPTSATTKKNSSRRNAWGNLSYADLITQAITSSQEKRLTLSQIYEWMVQNVPYFKDKGDSNSSAGWKNSIRHNLSLHNRFMRVQNEGTGKSSWWMINPDAKPGKSVRRRALSMETSKTEKRRGRVKKKPDALRNGVTADATPSPSSSISESLDIFPDSPIHSSFQLSPDFRQRASSNASSCSRLSPIPSLIPTEPEWSTDYTPSGDFASTSDFSPADYTQDQDLAGSLADSMKLHGADPFLNTYVPTTSSSSSGGSYRFTYGACPRHPHGGCACAPLYPAHPAHPTHSHQHSLDHFVRPPPPADPADIMQTENSQTQMVATSDATLMNGGIMVQTGPMGPTTVMGQIMGALNTGLSEDLNIEALEHSFDCNVDEVIRHELSMDGTLDFNFPQQNTAMAAEAESQFRAPSAPVPTTLSGGGASQTPYTVAPSWVH.TOR: MYMFLLKGHEDLRQDERVMQLFGLVNTLLQADPDTFRRDLVIQRYAVIPLSTNSGLIGWVPHCDTLHSLIKDYREKRKILLNIEHRIMQRMASDLEKLMLMQKVEVFEHALEHTAGDDLAKLLWLKSPSSEVWFERRTNYTRSLAVMSMVGYILGLGDRHPSNIMLDRVTGKFLHIDFGDCFEVAVTRDKFPEKIPFRLTRMLINAMEVTGIEGTYRRTCESVMEVLHRHRDSVMAVLEAFVYDPLLNWRLIDAGRRAGAPDDDGDASPQRPDDAPAETNLNKRALAIVNRVRDKLTGRDFTHIEENSVSVQRQVDLLIQQATSNENLCQCYVGWCPFW.Whole-mount adipose tissue immunostainingοC until mounted on slides.Females were sacrificed by crushing the ventral nerve and their abdomens were immediately dissected in cold phosphate buffered saline (PBS) to obtain fat body. Fat bodies were placed in 1.5ml microfuge tubes that were previously coated with 3% bovine serum albumin (BSA). Fat bodies were fixed in 1ml of 4% paraformaldehyde diluted in cold PBS (PFA) for 30 minutes at room temperature, rocking on a nutator. The PFA was then removed, and samples were rinsed twice with 1ml of PBS containing either 0.1% Triton X-100 (PBTX) or Tween 20 (PBTW) as a detergent. After rinsing, samples were washed three times with 1ml of 0.1% PBTX or PBTW, rocking each time for 15min on a nutator at room temperature. After the last wash, samples were incubated in 1ml of a blocking solution (0.1% PBTX or PBTW in 5% BSA) overnight at room temperature on a nutator. Samples were incubated in the indicated primary antibody concentration, diluted in blocking solution, overnight at room temperature. After washing three times for 15 minutes with PBTX or PBTW, samples were incubated in goat anti-rabbit AlexaFluor 488 secondary antibody diluted 1:250 in blocking solution and protected from light by covering with foil. Samples were then washed three times for 15 minutes with 0.1% PBTX or PBTW, protected from light. Following removal of wash solution, two drops of VectaShield containing DAPI were added. Samples were stored at 41X PBS – made from 10X PBS 3% BSA – made from 30% BSA 4% PFA – made from 16% PFA Triton X-100 - VWR, 0694-1LTween 20 – VWR, 0777-1LBlocking solution – 0.1% PBTX or PBTW and 5% BSA in 1XPBSRabbit anti-FOXO – GenScriptRabbit anti-TOR – GenScriptGoat anti-rabbit IgG AlexaFluor 488 – Invitrogen, A27034


Caenorhabditis elegans synthetic lethality screening. We identified the bromodomain protein, BET‐1, as a key regulator of actin function and longevity. Overexpression of bet‐1 preserves actin function at late age and promotes life span and healthspan in C. elegans. These beneficial effects are mediated through actin preservation by the transcriptional regulator function of BET‐1. Together, our discovery assigns a key role for BET‐1 in cytoskeletal health, highlighting regulatory cellular networks promoting cytoskeletal homeostasis.The actin cytoskeleton is a three‐dimensional scaffold of proteins that is a regulatory, energyconsuming network with dynamic properties to shape the structure and function of the cell. Proper actin function is required for many cellular pathways, including cell division, autophagy, chaperone function, endocytosis, and exocytosis. Deterioration of these processes manifests during aging and exposure to stress, which is in part due to the breakdown of the actin cytoskeleton. However, the regulatory mechanisms involved in preservation of cytoskeletal form and function are not well‐understood. Here, we performed a multipronged, cross‐organismal screen combining a whole‐genome CRISPR‐Cas9 screen in human fibroblasts with in vivo  Caenorhabditis elegans (2), we identified BET‐1/BRD4 as a novel regulator of the actin cytoskeleton (3). BET‐1/BRD4 promotes actin filament stability in C. elegans and senescent cells, which has direct impact on organismal health, survival, and life span.Using a cross‐species screening approach combining genome‐wide CRISPR‐Cas9 screening in human fibroblasts (1) and synthetic lethality screening in  ACSRactin cytoskeletal stress responseEVempty vectorGOgene ontologyRNAiribonucleic acid interference1Caenorhabditis elegans  , mediated by the heat‐shock transcription factor, HSF‐1. HSF‐1 is activated under thermal stress and promotes protein homeostasis through the upregulation of chaperones and other genes related to protein quality control , which promotes actin function, longevity, and healthspan in a multicellular eukaryote.To identify previously unidentified regulators of actin function, we adopted a multipronged, cross‐organismal screening approach. Since actin is one of the most highly conserved proteins, both in terms of sequence and functional homology, we held the rationale that an evolutionary‐conserved perspective could provide a powerful method to identify key regulators of actin function. We first utilized a CRISPR/Cas9‐driven growth‐based genetic screen in human cells to identify genes required for survival under actin stress. Actin stress was applied by exposure to cytochalasin D, a drug that inhibits actin polymerization by binding to F‐actin filaments and preventing the addition of actin monomers   and exhibits visible perturbations of actin function without affecting whole organismal physiology. Animals treated with 10% act‐1 RNAi develop normally to adulthood but display notable perturbations of actin quality in muscle cells  Figure . Importas Figure . Therefobet‐1, egrh‐1 (Early Growth factor Response factor Homolog), F53B6.5, and ikb‐1 (I Kappa B homolog) resulted in delayed development when combined with 10% act‐1 RNAi but exhibit no physiological phenotypes when knocked down alone. RNAi knockdown of ZK512.4 did not exhibit a developmental defect but showed sterility (no eggs were visible on the plate at day 1 of adulthood) when combined with 10% act‐1 RNAi, despite showing visible progeny formation when knocked down alone. ZK512.4 is a predicted ortholog of human SRP9 , a critical part of protein targeting to the endoplasmic reticulum , a major transcription factor that has been implicated in multiple diseases including cancer , which removes methionine residues from nascent polypeptide chains and are also implicated in cancer  for 24 h to induce senescence , the heat‐shock response (HSR), or oxidative stress response. We did observe a mild activation of genes involved in the endoplasmic reticulum UPR (UPRER). These data were further verified by direct comparison with a dataset identifying targets of the UPRER transcription factor, XBP‐1    Figure . Consist6 Figure , which e1 Figure , suggest2.4bet‐1B overexpressing worms was act‐3 , which has been previously linked to aging , another gene whose reduction has been linked to longevity , we opted for a secondary screening platform in bet‐1, the C. elegans ortholog of human BRD2 and BRD4. bet‐1 encodes a double bromodomain protein that has been originally characterized for its role in cell fate decisions  dosage has been previously linked to longevity in C57B6/J mice, although the underlying molecular basis was not identified , but may be detrimental where actin dynamics are required .What was intriguing was that BET‐1's impact on actin function is seemingly context dependent. We found that bet‐1 overexpressing animals, with act‐3 specifically being highly induced. We also observed changes in other cytoskeletal genes, including different regulatory factors. These genes showed opposing effects when compared to bet‐1 loss of function animals and were largely dependent on the HAT, mys‐1. Importantly, the transcriptional changes induced by BET‐1B were largely distinct from the UPRMT, HSR, and the oxidative stress response.Since we see that all visible BET‐1B protein is found within the nucleus and localizes to puncta which in no way resembles actin filaments, we believe that BET‐1B does not impact actin through direct interactions. Moreover, the beneficial effects of BET‐1B on actin and life span are dependent on MYS HATs, which acetylate histones at specific lysine residues, allowing BET‐1B binding to alter gene expression , which can impact organismal health. An exciting area of future work is to investigate whether a BET‐1B driven ACSR – possibly in coordination of other cytoskeletal regulators including DAF‐16 and HSF‐1 – can drive overall stress resilience and longevity.However, we did observe a mild overlap with UPRbet‐1B overexpression was dependent on the insulin/IGF‐1 transcription factor, DAF‐16. While DAF‐16 itself has not been directly tied to cytoskeletal function, FOXO transcription factors have direct impacts on actin function in mammalian systems  expressing hTERT and Cas9 were used for the CRISPR‐Cas9 based screen as previously described  and live/dead Aqua  staining was performed as per manufacturer's protocol. Briefly, cells were stained with both dyes for 20 min at room temperature in Annexin V binding buffer . Dyes were washed once with Annexin V binding buffer, resuspended with Annexin V binding buffer, and flow cytometry  was used to quantify staining. For statistical analysis, two‐way ANOVA with Dunnett's test was used to compare the ratio of live cells between the 0 μM control against each drug‐treated group. 3 technical replicates of 2 biological replicates were performed.For Annexin V staining, BJ fibroblasts were treated with cytochalasin D at 0–10 μM concentrations for 24 h and 3‐h treatment with 30 mM H4.2Human primary IMR‐90 fibroblasts were grown in DMEM, supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine at 37°C in a humidified atmosphere with 5% CO2 and 3.5% oxygen  per 100 μl culture medium. The old medium from senescent IMR‐90 cells was aspirated and 120 μl MTS assay solution per well was added. After 2 h incubation, 100 μl MTS assay solution aspirated from the plate in a fresh 96‐well clear plate and the absorbance at 495 nm was taken immediately using Microplate Reader.To examine the adhesion capacity of senescent cells after drug treatments, VybrantTM Cell Adhesion Assay Kit  was used according to the manufacturer's protocol with modification. Briefly, 30–40% confluent IMR‐90 cells were treated with etoposide and BET inhibitor/degraders drugs in 6‐wells plates as previously mentioned. After 8 days, cells from each well were trypsinized and pelleted down after neutralizing the trypsin with complete media. The cells were washed once with PBS and resuspended in 1 ml serum free DMEM containing 5 μM Calcein AM solution for 30 min. Then cells were washed twice with complete media to remove the excess Calcein AM. Finally, the Calcein AM labeled cells were suspended in 700 μl complete media and 100 μl of suspension was seeded per well in a PhenoPlate‐96 (PerkinElmer). Cells from each treatment were seeded in total 6 wells of 96 wells. After 4 h incubation, 3 wells from each treatment were washed 3 times with PBS to assay the adhering cells and 3 wells were maintained without washing to assay the total cells. Fluorescence was measured using Clariostar Multi‐Mode Microplate Reader using FITC channel. The percentage of adhered cells were calculated from the fluorescence of adhered cells against total cells seeded after background subtraction.To detect the level of actin polymerization, we used Alexa Fluor 488 Phalloidin , because this bicyclic peptide specifically binds to polymerized F‐actin. Approximately 12,000 IMR90 cells were seeded in a PhenoPlate‐96 (PerkinElmer) and treated with etoposide and drugs, as previously mentioned. After 8 days of etoposide treatment, cells were fixed with 4% paraformaldehyde. After washing the fixed cells with PBS three times, cells were permeabilized with 0.2% Triton‐X and blocked with 1% BSA solution in PBS for 30 min to reduce the background staining. After blocking, F‐Actin of the cells was stained with 165 nM Alexa Fluor 488 Phalloidin for 1 h at room temperature. Cells were washed with PBS three times and nuclei were stained with DAPI. The fluorescence of the wells was measured by using Clariostar Multi‐Mode Microplate Reader.4.3E. coli B strain and switched to growth at 20°C on HT115 E. coli K strain for all experimentation. HT115 bacteria carrying a pL4440 empty vector control or expressing double‐stranded RNA containing the sequence against a specific target gene were used for all experimentation. Experiments are performed on age‐matched animals synchronized using a standard bleaching protocol  and bleached in 1.8% sodium hypochlorite and 0.375 M KOH diluted in M9 until all carcasses were digested. Intact eggs were then washed 4× with M9 solution followed by L1 synchronization by floating eggs in M9 overnight in a 20°C incubator on a rotator for a maximum of 16 h. Synchronized animals are always grown on standard RNAi plates .All strains used in this study are derivatives of the N2 wild‐type worm from the Caenorhabditis Genetics Center (CGC) and are listed below. Worms are maintained at 15°C on OP50 bet‐1 overexpression, isoforms A, B, and C were defined as per  as a co‐injection marker, and 100 ng/μl of pD64 vehicle as filler DNA. Worms positive for the fluorescent pharynx were selected to identify stable arrays. Integration was performed by gamma irradiation where L4 worms were irradiated with 4000–4400 rems of radiation and integration events were selected by finding animals that maintained 100% frequency of co‐injection marker in the F3 generation. Lines were then backcrossed into N2 a minimum of 8x to eliminate mutations. For overexpression of 3xHA::GFP::bet‐1B, a GFP sequence containing introns and a 3xHA cassette was cloned upstream of the bet‐1B coding sequence. Injection and integrations were performed by SUNY Biotech.For bet‐1(uth41) mutant line, we used a Cas9‐RNA protocol as published on the IDT website via the Dernberg lab. Briefly, a mixture of 15 μM trRNA, and 22 μM crRNA (accatgggcaagtccgcgac) were incubated for 5 min at 95°C, cooled, then mixed with 24 μM Cas9 protein and left at room temperature for 5°C. 2.5 ng/μl of pEK2 (myo‐2p::tdtomato) was added as a co‐injection marker and this mixture was injected into C. elegans gonads. All progeny positive for the co‐injection marker were selected, then sequenced for INDELs that incorporated a premature stop codon. The bet‐1(uth41) mutant has a premature stop codon at amino acid 17.To synthesize the 4.4ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACC GGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTATGAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGC CATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGC GATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTTCAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAATCCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGA AAGAAAAAGAAAAACTTCAATATGTCCGAATCCTCGGATGATGAGACGTCGAATAGTCGAAAACGTCGAAAGAGAGAGAGCAGTGAATCACAGAGCTCTTCGTCCAGTGATGATGATTCAGATGATGAGGATAGGCCGAGTATTCCCCGTAAATCAGGTCAACCACCATCAACATCACGTGAATGGAATCAATCATCAGCTCCTCCACCACGAATGGGAGGAATGGGAGGACAACCACCAATGTCACGAGTACCTGCATCATCATCCACATCTGTATCAGCAATCGGAAAGAACAACGCAGCCGCCTCGTCGAATTCATATCAAGCTCCAAAACCTGCACCAGTACCAGCACCAACATCATCAAGACCTCCGGCAGCACCGAGACCACCGTCAAAACCAAAGAAAACGGGTGGAGCGAGTATTCTTGATACTCTACTTCCAGATACATTTGGAGCATCACCTCCCCAGTTTTTCCAGTCGCAACCAACAACGTCGGCTACGATTAGATCACCAACGGAAAGCCAACCCGGGAATGGTGAAGACGAGCAGACCAGGATTCAGAGGATGCGGATGGAGGCAAAGCGAGCCCGCCAAAAAGAAGACGAAGGCAGTGTCTCGTTGTCAAACCAAATGGAAATGATGGCTGCATTTGAATTTGATAATACATATTAA4.5ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACC GGCAAAAAGTGACGGTGGATCAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTATGAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGCCATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGCGATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTT CAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAAT CCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGA AAGAAAAAGAAAAACTTCAATATGTCCGAATCCTCGGATGATGAGACGTCGAATAGTCGAAAACGTCGAAAGAGAGAGAGCAGTGAATCACAGAGCTCTTCGTCCAGTGATGATGATTCAGATGATGAGGATAGGCCGAGTATTCCCCGTAAATCAGGTCAACCACCATCAACATCACGTGAATGGAATCAATCATCAGCTCCTCCACCACGAATGGGAGGAATGGGAGGACAACCACCAATGTCACGAGTACCTGCATCATCATCCACATCTGTATCAGCAATCGGAAAGAACAACGCAGCCGCCTCGTCGAATTCATATCAAAAATTTTATAATTGTTTTCACAGTTATACTCCACCTTTAAAAGbet‐1CTTGAAAAAAAAATCATCAAATTACTGGTAAATTTTTGTTAA ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACCGGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTAT GAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGCCATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGCGATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTTCAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAAT CCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGAAAGAAAATAA4.6CACAGGTCTCTAGTGTATCCACTTCGAATGCGATGCCCGAAACCTCTTCATCCATCCGTCTCCTTCTCGCTCTCTCTCTCTCTCTCTCTTCTCCATCTCTCTCCACATTTTGCCTGCTATCTCGTGATTGTCGTCCCGTCCGTGTTCCGCCGCACACACTGCCTGTCTTCTCTTAACCGTGTGTCGATCAACTCCCAAACCGCTACGCTATTTCTCTCTCCCTCTCTCTCTCTCTTCGGCGGTGACATTTCTGACTAGATGGTCATACAAAACGCGTGCTGCGCGCGCGCTCCGCAAAAATCGACGCGAATCGATTAATGTGCGTCTCGTTTCTCTATCTCTGACCGCCCCCGCTTCAACCTAACACTATTTTTGAATGCTTTTCAACTGTAACTTGCAGCTAATTAGAAGTTGAGAGATAACCTGTTGCGATTGGCTCCGGGCAAGGGTTGGGAGGTCGCACCAGAAATTTTAGAGCTCTAGGATTTCAAATTTTTGGGTTTCAAGACCGTAACATGATTTTCTTGGAAATTTATCACAAATCATGTAGAAAATCGATATCAGTAAGAGGGAGTGAGTGATCTATCATTTTTTATCTTTCGATCTGAAATTCCACAGCGAAGGTTTTCTGCCGAAATTTCGAAATTGGTATTTTGAACTATCCGATAATTCGTAGAACATCAAGATAAAGTGTCAACCTATAGAAAATCACATGATTCGTCAGAAAATAACATTAATTTCATATGAAATAGTTGAGAAAGTGCTCAAAAATGGCCTAAAATTATCCAATAATCGACATTTGACAACTTTCAGCACACTTTTGAACCGTTTATCAATTGTTTCTGCTGAAATAGACGTATTTTTCGGACGAATCGAGTGATTTCCTATAGTTTTACACTGATTTTTGACAAAAAAATATTGATAGAACATGGTGCATTAGGCAATTTTTTAGAATTGCCGTCTACACCTGATTTCGATGGGTCCTCGTGACAAGACCCAAAATTTTATTATTTTTATCGTTGAAAAAAATCAAATCAATAACACCGCAATCAC CATTTGCAAAGTTTAATTAAATACAATTTTTATTAAAATATTTCAGGAATAAAAATATTAGTCAGAATAATCCCATGTTCTTCTAGCGATTTCAACTAATTCTTTGAAAATAACATTTCTTGGAATT TAAGAATACGAAAATAGTCACTTCTTTGTATTCTAGAAACGCTAATTCCTGCAACCGACAAA TTAAAAGTACAAAAAATGATACGGCAAGCGCGCTCCAATTCAAATCGAGTCTCCCGCCTTCCTTGACGTCATTGCTAACAGCTGCTTCGGTTTTTTCCTCCAAATTTCGTGGTTCAAATTTTAT TTTTAATTGAATTTTAACAAAATAGGAAGCTAGTTGAGTAACATTTATTATTAATTTTGTAAAATATTCTGCAAATTCGGCGTTTTCTTTTAATTCAAATAAAAGTTTTCAATAAAAAAAATCGATATTTTCAG4.7CATCTCGCGCCCGTGCCTCTGACTTCTAAGTCCAATTACTCTTCAACATCCCTACATGCTCTTTCTCCCTGTGCTCCCACCCCCTATTTTTGTTATTATCAAAAAACTTCTCTTAATTTCTTTGTTTTTTAGCTTCTTTTAAGTCACCTCTAACAATGAAATTGTGTAGATTCAAAAATAGAATTAATTCGTAATAAAAAGTCGAAAAAAATTGTGCTCCCTCCCCCCATTAATAATAATTCTATCCCAAAATCTACACAATGTTCTGTGTACACTTCTTATGTTTTTTACTTCTGATAAATTTTTTTGAAACATCATAGAAAAAACCGCACACAAAATACCTTATCATATGTTACGTTTCAGTTTATGACCGCAATTTTTATTTCTTCGCACGTCTGGGCCTCTCATGACGTCAAATCATGCTCATCGTGAAAAAGTTTTGGAGTATTTTTGGAATTTTTCAATCAAGTGAAAGTTTATGAAATTAATTTTCCTGCTTTTGCTTTTTGGGGTTTCCCCTATTGTTTGTCAAGATTTCGAGGACGGCGTTTTTCTTGCTAAAATCACAAGTATTGATGAGCACGATGCAAGAAAGATCGGAAGAAGGTTTGGGTTTGAGGCTCA GTGGAAG4.8ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatac taactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTGTTATGGTGTTCAATGCTTCTCGAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTAT GTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcgg tactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGA ATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATA CAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA4.9TATCCATATGACGTGCCGGACTACGCGTACCCGTATGATGTTCCAGACTACGCCTATCCGTACGACGTACCAGATTATGCA4.10CAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGCGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACCGGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGCAGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCG4.11AAAAAAGCAGGCTTGACCGAGCCGAAGAAGGAGATTATAGAGGACGAAAATCATGGAATAT CCAAGAAAATACCAACAGATCCCAGGCAATACGAGAAAGTTACAGAGGGATGCCGGTTATTGGTCATGATGGCTTCACAAGAAGAAGAAAGATGGGCCGAAGTTATTTCAAGATGCCGAGCTGCAAATGGTTCAATTAAATTCTATGTCCATTATATCGATTGCAACCGAAGACTTGACGAATGGGTTCAGTCTGATAGGCTCAATTTAGCGTCGTGTGAGCTACCAAAAAAAGGAGGAAAGAAAGGAGCACACTTGCGGGAAGAAAATCGAGATTCGAATGAAAATGAAGGAAAGAAAAGCGGCCGAAAACGAAAGATTCCACTACTTCCGATGGATGATCTCAAGGCGGAATCCGTAGATCCATTACAAGCAATTTCAACGATGACCAGCGGATCTACTCCAAGTCTTCGAGGTTCCATGTCGATGGTCGGCCATAGTGAAGATGCAATGACAAGGATCCGAAATGTCGAATGCATTGAACTAGGAAGATCACGAATTCAGCCATGGTACTTTGCACCTTATCCACAACAATTGACAAGTTTGGATTGTATTTATATTTGCGAATTTTGTCTGAAATATCTAAAGTCGAAAACTTGTCTGAAACGGCACNTGGAAAAATGTGCAATGTGTCACCCACCTGGCAATCAAATCTACAGTCACGATAAACTTTCATTTTTTGAAATCGACGGCCGCAAAAACAAAAGCTATGCTCAGAATCTATGCCTGCTTGCCA AACTT4.12C. elegans orthologs of human genes from the cytochalasin screen were identified using Ortholist 2 , images were acquired on either a Zeiss AxioObserver.Z1 microscope equipped with a lumencor sola light engine and Ziess axiocam 506 camera driven by Zeiss ZenBlue software using a 63x/1.4 PlanApochromat objective, standard dSRed filter (Zeiss filter set 43), and a DFC9000 camera; or a Leica Thunder Imager equipped with a 63x/1.4 Plan AproChromat objective, standard dsRed filter (11525309), Leica DFC9000 GT camera, a Leica LED5 light source, and run on LAS X software. For confocal microscopy (gly‐19p::LifeAct), imaging was performed on a Stellaris 5 confocal microscope equipped with a white light laser source and spectral filters, HyD detectors, 63x/1.4 Plan ApoChromat objective, and run on LAS X software.For high magnification live‐cell imaging, animals are picked off of plates and mounted directly onto a microscope slide containing M9 + 0.1 M sodium azide. For standard wide‐field microscopy  floating in M9 in a rotator at 20°C, then washed 3x with M9 and plated on standard OP50 plates for 24 h, then imaged.For imaging of fluorescent transcriptional reporters, animals were synchronized via bleaching and grown on standard RNAi plates and imaged at day 1 of adulthood. For imaging, animals were moved onto standard NGM plates without bacteria containing 5 μl of 100 mM sodium azide to paralyze worms. Paralyzed worms were lined up and imaged immediately on a Leica M205FCA automated fluorescent stereoscope equipped with a standard GFP filter and Leica K5 camera and run on LAS X software. 3 biological replicates were performed per experiment with 4.14t‐test using Prism software.To measure gut bacteria invasion, animals were synchronized via bleaching and plated from hatch on RNAi of choice mixed with 20% HT115 bacteria expressing mCherry as previously described . The aqueous phase was mixed 1:1 with isopropanol then applied to a Qiagen RNeasy Mini Kit (74106) and RNA purification was performed as per manufacturer's directions.bet‐1B overexpression, bet‐1 RNAi, and bet‐1(uth41) mutants were compared to N2 wild‐type control. In addition, bet‐1B overexpressing worms were grown on mys‐1 RNAi and compared to a mys‐1 RNAi control.Library preparation was performed using a Kapa Biosystems mRNA Hyper Prep Kit sequencing was performed at the Vincent J Coates Genomic Sequencing Core at the University of California, Berkeley using an Illumina HS4000 mode SR100. Four biological replicates were measured per condition. Reads per gene were quantified using kallisto  using 1 μg of RNA. RT‐PCR was performed using NEB Q5 DNA polymerase as per manufacturer's guidelines using primers listed below. Four biological replicates were performed per condition. Image quantification was performed using ImageJ by drawing an ROI 974 of equal size around each band and quantifying for integrated density. Data was normalized to a 975 4.16Thrashing assays were performed on animals synchronized via bleaching and aged on plates containing FUDR from day 1. 100 μl of 10 mg/ml FUDR were spotted on the bacterial lawn. At the desired age, plates containing adult animals were flooded with 100 μl of M9 solution, and 30 sec videos were acquired on an M205FCA stereomicroscope equipped with a Leica K5 microscope and run on LAS X software. Thrashing was measured by eye over a 10 s period. A single trash is defined as bending of >50% of the animal's body in the opposite direction. Representative data of three independent biological replicates are presented. Dot plots were generated using Prism 7 software where every dot represents a single animal and lines represent median and interquartile range. All statistics were performed using nonparametric Mann–Whitney testing.4.17A synchronized population of animals were collected via bleaching and 10 L4 animals were moved onto individual plates. Every 12 h, animals were moved onto fresh plates and plates containing eggs were stored in a 15°C incubator for 2–3 days. All live progeny on every egg‐lay plate were scored and summed to determine brood size. Dot plots were generated using Prism 7 software where every dot represents a single animal and lines represent median and interquartile range. All statistics were performed using non‐parametric Mann–Whitney testing.4.18Caenorhabditis elegans life span assays were performed on standard RNAi plates and were all performed at 20°C as previously described (Bar‐Ziv et al., Caenorhabditis elegans experiments for manuscript revision. N.Dasgupta performed all senescent cell culture experiments. N.Dutta performed RTPCR analysis and assisted with life spans. H.Z., and W.F. performed all standard cell culture experiments. C.K.T., E.A.M., and O.S. performed all computational analysis for CRISPR‐Cas9 screening. D.M., A.A., S.H., and T.C.T. assisted with C. elegans experiments. P.D.A. supervised all senescent cell culture experiments. M.A.T. performed essential experiments that assisted in development of the manuscript. All authors edited the manuscript.G.G. and R.H.S. designed all experiments, performed or oversaw all experiments, and prepared the figures and manuscript. R.B.Z. performed computational analysis, figure construction, and writing for all transcriptomics data. M.A. performed all All authors of the manuscript declare that they have no competing interests.Figures S1–S9Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.Table S8Click here for additional data file.


Titin is the largest protein in humans, composed of more than one hundred immunoglobulin (Ig) domains, and plays a critical role in muscle’s passive elasticity. Thus, the molecular design of this giant polyprotein is responsible for its mechanical function. Interestingly, most of these Ig domains are connected directly with very few interdomain residues/linker, which suggests such a design is necessary for its mechanical stability. To understand this design, we chose six representative Ig domains in titin and added nine glycine residues (9G) as an artificial interdomain linker between these Ig domains. We measured their mechanical stabilities using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) and compared them to the natural sequence. The AFM results showed that the linker affected the mechanical stability of Ig domains. The linker mostly reduces its mechanical stability to a moderate extent, but the opposite situation can happen. Thus, this effect is very complex and may depend on each particular domain’s property. The giant muscle protein titin is a tandem modular construction designed polyprotein containing more than two hundred individually folded domains, such as immunoglobulin-like (Ig) and fibronectin-type III domains . These dTo examine the linker effect, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the mechanical stability of human Ig domains. SMFS can manipulate a single molecule mechanically ,20,21,22Thus, we choose six consecutive Ig domains of titin, including I27, I28, I29, I30, I31, and I32, as a representative unit (I27–I31), to study the interdomain linker effect b. The stA high-precision AFM measurement system has been used for accurate measurement and comparison of Ig domains in titin ,65,66,67n = 1148, Thus, we built polyprotein Coh-I(27–32)/9G-NGL with a 9G linker for measurement. The 9G linker is present between each Ig domain except for the two end I27 and I32 domains a. By appn = 1808, Then, we used polyprotein Coh-I(27–32)-NGL with natural sequence for AFM measurement and comparison. The same cantilever used previously for the polyprotein with the linker was used here again to minimize the error. As expected, a similar unfolding pattern was observed b,d,e. Hon = 860). Then, AFM measurement on Coh-I(28–30)-NGL with natural sequence showed a force of 325 ± 35 pN (n = 965).To confirm this effect, we chose three Ig domains only and constructed two shorter polyproteins, Coh-I(28–30)/9G-NGL and Coh-I(30–32)/9G-NGL, for measurement. Indeed, stretching these polyproteins resulted in a shorter force-extension curve with only two 28 nm-peaks from I28, I29, or I31, I32, and one 11 nm-peak from I30, as expected . For Cohn = 1120). However, AFM measurement on the natural sequence showed a different result. Two peaks were observed in the histogram, with a force of 276 pN and 345 pN, respectively (n = 1804), which has not been observed before. For Coh-I(30–32)/9G-NGL, the histogram of unfolding force from I31 and I32 showed a single peak with an average force of 320 ± 33 pN , and 186 ± 55 pN (n = 225) without linker. For I(28–30), 9G linker is only present on the N terminus. The force was 197 pN (n = 570), and 172 ± 24 pN (n = 910) without linker. Finally, for I(30–32), 9G linker is only present on the C terminus. The force was 159 ± 23 pN (n = 477), and 175 ± 24 pN (n = 404) without the linker /9G-NGL, Coh-I(27–32)-NGL, Coh-I(28–30)/9G-NGL, Coh-I(28–30)-NGL, Coh-I(30–32)/9G-NGL, Coh-I(30–32)-NGL were obtained after Gibson assembly-based method [Escherichia coli strain BL21 (DE3) and cells were cultured overnight in LB medium at 18 °C by the addition of 1mM IPTG. The cells were pelleted by centrifugation and the polyprotein purification by Ni-NTA affinity. After using wash buffer  to purify the target proteins, the polyproteins were eluted in elution buffer . Protein ligase AEP was obtained according to literature [d method . All platerature .Protein immobilization: The glass coverslips  and probes  were cleaned by plasma. Then, both probes and coverslips were immersed in 1% (v/v) APTES toluene solution for 1 h to add the NH2 group, followed by a reaction with Milli-Q water containing 2 mM ImSO2N3, 4 mM K2CO3, and 20 mM CuSO4 to add the N3 group. After flushing, they were further reacted with DBCO-PEG4-maleimide for at least 2 h to add the maleimide group. Peptide C-ELP20-NGL and GL-ELP20-C were respectively reacted onto the probe and coverslip. For AFM-SMFS measurement, 50 μL AFM buffer  containing 100 μM Ig proteins and 50 μM AEP were pipetted on the glass slides for 40 min. The cantilevers were incubated with 50 μL solution of 60 μM GL-CBM-XDoc and 50 Μm AEP in the AFM buffer.AFM-SMFS Experiment: Measurements using Coh-Doc interaction of high rupture force as a standard were carried out Nanowizard4 (JPK) atomic force microscope. Using the equipartition theorem, the spring constant of ~30 pN nm−1 was obtained by calibrating the MLCT-Bio-DC (Bruker) cantilever in the AFM buffer solution. The functionalized cantilevers and glass coverslip immobilize polyproteins in AFM buffer at pH 7.4. All AFM experiments were performed at a constant pulling speed of 1000 nm·s−1.9G-I28-9G-I29-9G-I30-9G-I31-9G-I32)Protein sequence (I27-GGGGGGGGGPLIFITPLSDVKVFEKDEAKFECEVSREPKTFRWLKGTQEITGDDRFELIKDGTKHSMVIKSAAFEDEAKYMFEAEDKHTSGKLIIEGIGGGGGGGGGRLKFLTPLKDVTAKEKESAVFTVELSHDNIRVKWFKNDQRLHTTRSVSMQDEGKTHSITFKDLSIDDTSQIRVEAMGMSSEAKLTVLEGGGGGGGGGGDPYFTGKLQDYTGVEKDEVILQCEISKADAPVKWFKDGKEIKPSKNAVIKTDGKKRMLILKKALKSDIGQYTCDCGTDKTSGKLDIEDRGGGGGGGGGEIKLVRPLHSVEVMETETARFETEISEDDIHANWKLKGEALLQTPDCEIKEEGKIHSLVLHNCRLDQTGGVDFQAANVKSSAHLRVKPRGGGGGGGGGVIGLLRPLKDVTVTAGETATFDCELSYEDIPVEWYLKGKKLEPSDKVVPRSEGKVHTLTLRDVKLEDAGEVQLTAKDFKTHANLFVKEPLIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLAASPDCEIIEDGKKHILILHNCQLGMTGEVSFQAANTKSAANLKVKEL


The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Using ONE-seq, we discover more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveal epitranscriptome-wide dynamics of NAD-RNAs with age. ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification. In SPAA (SPAAC) . As a re ethyl]-biotinamide, CAS: 717119-80-7, 5 mM, Amatek scientific, catalog: B-1328) under the catalysis of ADPRC  in 100 μl of ADPRC reaction buffer   was reacted with HEEB (4HCO3 in water and (B) acetonitrile at a flow rate of 0.4 ml/min. The injection volume was 2 μl. The gradient was as follows: 0 min 0% B, 2 min 0% B, 4 min 20% B, 17 min 40% B, 19 min 100% B, 24 min 100% B, and 30 min 0% B. The retention time of NAD and ADPR-Biotin (product) was 9.393 min and 9.929 min, respectively. The corresponding compounds were purified from HPLC and then analyzed by LC–MS by Agilent Technologies 6120 Quadrupole with a C18 column . The mobile phase was composed of (A) 0.1% formic acid in water and (B) acetonitrile at a flow rate of 0.4 ml/min. The injection volume was 5 μl. The gradient was as follows: 0 min 10% B, 4 min 100% B, and 7 min 100% B.Samples were analyzed by an Agilent Technologies 1260 Infinity with a C18 column  monitoring at 260 nm. The mobile phase was composed of (A) 100 mM NHTAATACGACTCACTATTACTGTGTCGTCGTCGTCTGCTGTCTCTCTCTCGCGGGC-3′; boldface letters denote the sequence of T7 class II promotor (ϕ2.5)) and (anti-sense: 5′-GCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3′) ) and (anti-sense: 5′-GGCAGCAAGCCGGAGGAGCAGAAGAACGACAGGGCCAGCGAGCCGCAGGCGGCAGCGCGCACGCCCACGCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3′). To prepare spike-in ppp-RNA of 62 nt, oligonucleotide was synthesized (Genewiz) and annealed to make double-stranded DNA template ) and (anti-sense: 5′-TGGCTGGCCGCATGCCCGCGGCAGAAGTGACATTATGGCTAAAGTTGTGGTAGCACTTCCCTAATAGTGAGTCGTATTA-3′). To prepare spike-in NAD-RNA (500 nt) and m7Gppp-RNA (500 nt) with identical sequence, oligonucleotide without adenine was synthesized (Genewiz) and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5′-AAGAAGAACACCAAGAAGCCGAACAACAGCAAGACGGCCAAGAAAAAGACGAAGAGGCAGAAGAAGAAGAACACCAGCAAGAGCCCCAGGAAGAAGCCGACCACCAAGAAGACGAAGCCCAACAGCACGAAGCGGAACACCAGGGAGACGCCCACGAACAACACCACGGCGCGGGACAAGAAGAAGCCGACGACCAAGAAGAAGAAGGAGCGCACCAGGACGAAGCCAACGGGCAAGGCGGACAAGAAGAAGACGAGCAGCAACAAGAGGACGGGGAAGCGGCAGAAGCACAGCACGCCGAAGGACAGGGAGGACACGAGGGAGGGCCAGGGCACGGGCAGCAAGCCGGAGGAGCAGAAGAACAACAGGGACAGCAAGCCGAAGGAGGCAACGCCCACGCCCACGCCGGACACGCAGAACAAGAGGCCGAAAACGACGCCGACCAGCACGACCAGGAAGGGCACCACCCCGGAGAACAGCACCACGCCCAAGCACACCATAATAGTGAGTCGTATTA-3′). For in vitro transcription, 10 μM of double-stranded DNA (dsDNA) template in 100 μl transcription buffer , along with 1 mM of each of GTP, CTP and UTP, with 1mM ATP , 4 mM NAD (for NAD-RNA) or 4 mM m7GpppA  (for m7G-RNA), 10 μl of T7 RNA polymerase , 5% DMSO, 5 mM DTT and 2.5-unit RNase inhibitor were added and the transcription mixture was incubated at 37°C for 4 h. The reaction was incubated with 11-unit DNase I  at 37°C for 30 min to remove the DNA template. RNA was then extracted using acid phenol/chloroform and precipitated with isopropanol  at -80°C overnight. The RNA pellet was washed twice with 75% ethanol, air-dried, redissolved in DEPC-treated H2O, and stored at –80°C. For spike-in RNAs used as internal controls for gene-specific qRT-PCR assessment, NAD-RNA is 106 nt as described above. To prepare ppp-RNA, 88 nt oligo nucleotide was synthesized  and annealed to make double-stranded DNA template. Primer extension was used to increase the length of the template to encode 106 nt RNA ) and (anti-sense: 5′-CAAGGCGTTGGTCGCTTCCGGATTGTTTACATAACCGGACATAATCATAGGACCTCTCACACACAGTTCGCCTCTTTGATTAACGCCCAGCGTTTTCCCGGTATCCAATAGTGAGTCGTATTAGTGATC-3′). To prepare 45 nt m7Gppp-RNA, oligo nucleotide was synthesized (Genewiz) and annealed to make double-stranded DNA template. Primer extension was used to increase the length of the template to encode 45 nt RNA ) and (anti-sense: 5′-CGACAGGGCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3′).To prepare short RNA of 38 nucleotides (nt), 10 μM template oligo DNA (template sequence: 5′-GATCACGATC-3′)  were com7G-RNA (500 nt) and ppp-RNA (500 nt) spike-ins with different sequences from that of NAD-RNA . The synthesis of m7G-RNA (500 nt) was as described in  and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5′- TGGCCTTGTAGGTGGTCTTGACCTCAGCGTCGTAGTGGCCGCCGTCCTTCAGCTTCAGCCTCTGCTTGATCTCGCCCTTCAGGGCGCCGTCCTCGGGGTACATCCGCTCGGAGGAGGCCTCCCAGCCCATGGTCTTCTTCTGCATTACGGGGCCGTCGGAGGGGAAGTTGGTGCCGCGCAGCTTCACCTTGTAGATGAACTCGCCGTCCTGCAGGGAGGAGTCCTGGGTCACGGTCACCACGCCGCCGTCCTCGAAGTTCATCACGCGCTCCCACTTGAAGCCCTCGGGGAAGGACAGCTTCAAGTAGTCGGGGATGTCGGCGGGGTGCTTCACGTAGGCCTTGGAGCCGTACATGAACTGAGGGGACAGGATGTCCCAGGCGAAGGGCAGGGGGCCACCCTTGGTCACCTTCAGCTTGGCGGTCTGGGTGCCCTCGTAGGGGCGGCCCTCGCCCTCGCCCTCGATCTCGAACTCGTGGCCGTTCACGGAGCCCTCCATGTAATAGTGAGTCGTATTAGTGATC-3′). To prepare ppp-RNA, oligonucleotide was synthesized (Genewiz) and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5′-TCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATAATAGTGAGTCGTATTA-3′).For boronate affinity experiment, we synthesized mribed in . To prep2) at 37°C for 1 h. Product was purified with Zymo column  according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).NAD-RNA (1 μg) was reacted with 40 mM and 100 mM HEEB (1 M stock in DMSO) under the catalysis of ADPRC (25 μg/ml) with 0.4 U/μl of RNase Inhibitor  in 100 μl of ADPRC reaction buffer  were washed 3 times with immobilization buffer  , and the254nm (0.18 J/cm2) twice. Membrane was blocked in 5% BSA in PBST (0.1% Tween20 in PBS) for 1 h, followed by incubation with Alexa Fluor® 790 Streptavidin  at room temperature for 2 h in the dark. Membrane was washed with PBST and PBS for 3 times, respectively, and imaged on the Odyssey LiCor CLx scanner (Li-Cor Biosciences) with the software set to auto-detect the signal intensity at 800 nm channel. Finally, the membrane was stained with methylene blue solution (0.3% w/v methylene blue + 30% v/v ethanol + 70% v/v H2O)  which was then cross-linked by UVv/v H2O)  to visua7Gppp-RNA (45 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer  at 37°C for 1 h. 100 μl of DEPC-treated H2O was then added and acid phenol/ether extraction was performed to stop the reaction  and 0.4 U/μl of RNase Inhibitor  at 25°C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris–HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H2O. The biotinylated RNA on the beads was extracted with Trizol LS  and chloroform. Input (see above) RNAs and the biotinylated RNA on the beads were analyzed by an 8% polyacrylamide TBE urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).100 ng of spike-in NAD-RNA (38 nt) and 200 ng of mreaction . RNAs we7Gppp-RNA (38 nt) was performed with 1 μl of NudC  in 25 μl of NudC reaction buffer  at 37°C for 30 min. Product was purified with Trizol LS  and analyzed by an 8% polyacrylamide TBE–urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).De-capping of 200 ng NAD-RNA (38 nt) or m7Gppp-RNA (45 nt) were mixed together and incubated with 500 nM NudC  in 25 μl of NudC reaction buffer  at 37°C for 30 min. Product was purified with Trizol LS  and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).100 ng of spike-in NAD-RNA (38 nt) and 200 ng of m2O was then added and acid phenol/ether extraction was performed to stop the reaction . Total RNAs (100 μg) was incubated with 100 mM HEEB (1 M stock in DMSO), ADPRC  and 0.4 U/μl of RNase Inhibitor  in 100 μl of ADPRC reaction buffer at 37°C for 1 h. 100 μl of DEPC-treated Hreaction . RNAs we2O. To ensure complete elution, biotin-conjugated RNAs were replaced from streptavidin beads by incubating with 1 mM biotin buffer  at 94°C for 8 min, followed by incubation with 500 nM NudC  in 25 μl of NudC reaction buffer  at 37°C for 30 min. After NudC treatment, biotinylated-RNAs that are resistant to NudC catalysis, potentially derived from contaminating m7G-RNAs, were retained on beads by incubation with high-capacity streptavidin particle  at 25°C for 30 min. Eluted RNAs in the supernatant were used for next step.After HEEB reaction, biotinylated RNAs were incubated with streptavidin bead particles  and 0.4 U/μl of RNase Inhibitor  at 25°C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris–HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H7Gppp-RNA (38 nt) was performed with or without 1 μl of yDcpS  in 1X yDcpS reaction buffer  in 20 μl total volume at 37°C for 1 h. Product was purified with Zymo column  according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).De-capping of 200 ng NAD-RNA (38 nt) and m7Gppp-RNA (106 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with or without ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer  at 37°C for 1 h, followed by NudC-catalyzed NAD-RNA elution. Input (see above) and NudC-eluted RNAs from three biological replicates were used for qRT-PCR. Reverse transcription was performed with gene specific primers using SuperScript III SuperMix . qPCR was performed using SYBR Green master mix  to detect NAD-RNA, ppp-RNA or m7Gppp-RNA from three biological replicates using specific primers described in t test.Total RNAs (100 μg) and 100 ng of spike-in NAD-RNA (106 nt), ppp-RNA (106 nt) or mInput (see above) and NudC-eluted RNAs from four biological replicates of livers from 2- and 18-month old C57BL/6 mice were used for NGS library construction, in accordance with manufacturer's instructions . Library quality was assessed by Bioanalyzer 2100 , and quantification was performed by qRT-PCR with a reference to a standard library. Libraries were pooled together in equimolar amounts to a final 2 nM concentration and denatured with 0.1 M NaOH . Libraries were sequenced on the Illumina NovaSeq 6000 system .7Gppp-RNA, 1% NAD-RNA/99% m7Gppp-RNA, 5% NAD-RNA/95% m7Gppp-RNA or 10% NAD-RNA/90% m7Gppp-RNA, respectively. The mixture of total RNAs and spike-in RNA (500 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer  at 37°C for 1 h, followed by NudC-catalyzed NAD-RNA elution. Input (see above) and NudC-eluted RNAs from three biological replicates of livers from 18-month old C57BL/6 mice were subjected to polyA-selected RNA sequencing as mentioned above.Total RNAs (100 μg) were mixed with 1 ng of 3′-end polyadenylated spike-in RNA (500 nt) that had 0% NAD-RNA/100% m7Gppp-RNA (38 nt) was ligated with 5 μM 3′ adaptor oligo listed in 1 μg of NAD-RNA (38 nt) or mBoronic acid beads  were washed 3 times with binding buffer , and then incubated with RNA oligonucleotide in binding buffer in a thermomixer at 37°C for 2 h. Beads were then washed 3 times with wash buffer ; RNA oligonucleotide bound by the beads was extracted with Trizol LS ). Purified RNA product was analyzed by an 8% polyacrylamide TBE–urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).7Gppp-RNA, and 1 ng of ppp-RNA). RNAs were incubated with yDcpS  in 50 μl of 1X yDcpS reaction buffer at 37°C for 1 h, to remove m7G cap. After purification with Trizol LS , yDcpS-treated RNAs were combined with 100 μmol oligo d(T)30VN in 100 μl 0.5× SSC buffer , and incubated at 80°C for 5 min followed by 50°C for 60 min. Then, 1 μl RNase H  and 5.6 μl 10× Reaction buffer  were added. The mixture was incubated at 37°C for 20 min, and purified with Trizol LS . This step aims to promote 3′-end adaptor ligation by removing polyA sequence tract from the endogenous transcripts. Prior to 3′-end ligation, polyA-depleted RNAs were treated with 2 U CIAP  in the presence of 40 U RNaseOUT at 37°C for 1 h to remove 5′-terminal phosphate. RNA was extracted with Trizol LS . 5 μg of CIAP-treated RNA products were ligated with 100 μM 3′ adaptor oligo listed in 2O. Reverse transcription was performed with adaptor-based primer listed in 7G-RNA (500 nt) was used as a negative control. Primers were listed in t-test.Total RNAs were extracted from three biological replicates of livers from 2-month old C57BL/6 mice as described above. For each replicate, total RNAs (50 μg) were mixed with polyadenylated spike-in RNAs   with theDESeq2 (v1.30.0) . NAD-RNAs were defined as fold change of normalized transcript counts ≥2 and FDR <0.05 in NudC-treated samples compared to those in input samples. Gene annotation information, such as chromosome, gene-types, gene-lengths and intron coordinates were retrieved from Gencode (M23) annotations. The violin plot, bar plot, line chart and scatter plot were generated by R package ggplot2 (v3.3.2) (Genes that had zero count in more than 25% (3 out of 12) sequencing libraries were removed before performing differential analysis. Principle component analysis (PCA) was performed with R function ‘prcomp’ using a transformed counts matrix by function ‘vst’. To identify NAD-RNA from total RNA-seq data, we used R package v1.30.0)  to perfo(v3.3.2) .gprofiler2 (v0.2.1)  in Cytoscape (v3.8.2)  . Pathway(v0.2.1)  and the (v0.2.1)  without (v3.8.2) . Network(v3.8.2) .2) at 37°C for 1 h. 100 μl of DEPC-treated H2O was then added and acid phenol/ether extraction was performed to stop the reaction  following the manufacturer's protocol with primers listed in Total RNA was extracted from four biological replicates of livers from 2-month old C57BL/6 mice as described above. For each replicate, 100 μg total RNA, mixed with 1 ng of NAD-RNA (106 nt) and 1 ng of ppp-RNA (106 nt), was incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer  ethyl]-biotinamide) as a reactant, allowing only one reaction to biotinylate NAD-capped RNAs. To avoid contaminating signals, we designed a NudC-based post-treatment to elute biotin-conjugated RNAs specifically derived from NAD, but not m7G-capped transcripts from streptavidin beads. Therefore, our method circumvented laborious steps to purify mRNAs followed by clearance of m7G-capped RNAs. With significantly simplified procedures, our method enabled NAD-RNA profiling directly from total RNA. Based on the same platform, qRT-PCR analysis on specific NAD-RNAs could be readily applied.Compared to previous methods that require multiple reactions, we introduced HEEB  and NAD-RNA (38 nt) with 100 mM HEEB to the reaction. While NAD-RNA reacted with HEEB, as indicated by the presence of a biotinylated form, m7G-RNA had no such a product, reflecting specificity  but not m7Gppp-RNA (38 nt) as shown by a lower-sized band corresponding to the de-capped product  to the same reaction. The result clearly showed that NudC was able to selectively de-cap NAD-capped (38 nt) but not m7G-capped RNA (45 nt)  but not m7Gppp-capped forms (38 nt) from streptavidin beads , but not NAD-RNA (38 nt) was sensitive to yDcpS treatment, a decapping enzyme that hydrolyzes the triphosphate linkage of m7G-capped RNA  Figure . We tests Figure  and F. Apped RNA .7G-capped RNA (m7Gppp-RNA), respectively). HEEB reacted with NAD-RNA (106 nt) and m7Gppp-RNA (106 nt), but not ppp-RNA (106 nt), resulting in a band retained by the streptavidin beads (7Gppp-RNA (106 nt) could be detected on streptavidin beads; treatment of NudC, however, found no evidence to elute m7Gppp-RNA (106 nt)  Figure .7G-cap, followed by polyA tails. Presumably, endogenous transcripts may contain both NAD and m7G-capped forms, though the percentage may differ for particular genes. The rationale of this design is to mimic endogenous genes that have either low (0% or 1%) or relatively high (5% or 10%) NAD modification. Total RNAs were mixed with spike-ins that had different ratios of NAD-RNA and m7G-RNA. The sample that contained 100% m7G-RNA spike-in represented a gene with no NAD capping, which allowed the assessment of the specificity of ONE-seq platform. Other samples that contained 1%, 5%, and 10% of NAD- relative to m7G-RNA spike-ins were used to determine the capture sensitivity. We subjected 100 μg total RNA from mouse livers mixed with 1 ng spike-in RNA to ONE-seq experiment, followed by polyA-selected RNA sequencing. In the sample mixed with 100% m7G-RNA spike-in, we found no enrichment  but had either NAD or mt Figure . As demot Figure , represee Figure , suggeste Figure , reflect7G-capped RNAs.Fourth, we determined the noise-cancelling effect of NudC by comparative analysis of RNA-seq datasets. Total RNAs, after HEEB reaction, were captured by streptavidin beads, followed by either NudC-catalyzed elution (NudC+) or mock treatment (NudC-) . polyA-s7G and NAD-capping.We tested the utility of ONE-seq. We profiled NAD-RNAs from mouse liver tissues of young (2-month) and aged (18-month) cohorts. After quality control, we obtained in average ∼12.3 million high-quality and uniquely mapped sequencing read pairs from each library . Assessmcis diols, occurring naturally at the 3′-end of RNA, as well as in the 7-methylguanosine of m7G-cap and the nicotinamide riboside of NAD-cap  at the terminal is ligated to the 3′-end of RNA, such that the vicinal-diol moiety of the ribose at the 3′-end of RNA no longer exists. At this step, affinity binding can only occur between the boronyl group from boronic acid and 1,2-cis diols from the nicotinamide riboside of NAD-cap. Importantly, we perform reverse transcription using an adaptor-specific primer to harvest RNAs with 3′end blocker, thereby abolishing false signals from boronate binding of RNAs that are not properly ligated with adaptor. Consequently, only RNA transcripts that contain NAD-cap can be selectively enriched by boronate affinity.We validated newly identified NAD-RNAs by an ADPRC-independent method. To do this, we applied boronic acid beads. Our rationale lies at the fact that boronyl groups of boronic acid can form relatively stable complexes with 1,2- NAD-cap . Inspire NAD-cap ,38, we d NAD-cap . First, 7G, but not NAD, from RNA oligos (38 nt). In the presence of RNA ligase, 3′adaptor was efficiently ligated to the spike-in RNAs (38 nt), yielding an upper band in the gel  that were designed with different sequences. This experiment clearly demonstrated that NAD-RNA (500 nt) spike-ins, but not ppp-RNA (500 nt) and m7Gppp-RNA (500 nt), were selectively and significantly enriched by boronic acid beads  and transcription (e.g. RNA polymerase II) were found to contain NAD-cap. Also, transcripts with 5′-NAD modification were enriched in cell cycle (e.g. centromere protein and CDK) and DNA damage responses (e.g. ATM and TP53). Not just housekeeping genes, transcriptional factors (e.g. BMP5 and Interferon), epigenetic pathways , and histone-related genes were also subject to NAD modification Figure . In mitoIn line with an age-modulated decrease of NAD , both thAbove evidence supported the notion that ONE-seq platform permits epi-transcriptome-wide profiling directly from total RNA, prompting us to extend its application for gene-specific assessment by qRT-PCR. To do this, we included non-capped ppp-RNA (106 nt) as a baseline negative control, and NAD-RNA (106 nt) as a positive control. Total RNA and internal spike-in controls were subjected to ONE-seq experiment, followed by qRT-PCR. The relative abundance was calculated between NudC + and input samples Figure . Our datN-acetylglucosamine (UDP-GlcNAc), can be incorporated at the 5′-end of RNA during transcription initiation in both prokaryotes and eukaryotes , flavin adenine dinucleotide (FAD), uridine diphosphate glucose (UDP-Glc) and uridine diphosphate karyotes . CapZymeNCIN cap . As a re7G) has been found to contaminate the NAD-RNA profile. As such, SPAAC-NAD-seq , Mup13 (http://genome.ucsc.edu/s/lda97/Mup13_ONE-seq), Mup19 (http://genome.ucsc.edu/s/lda97/Mup19_ONE-seq) and Mettl7b (http://genome.ucsc.edu/s/lda97/Mettl7b_ONE-seq) can be accessed at UCSC Genome Browser online sessions. All source codes for data analysis are available at Zenodo https://doi.org/10.5281/zenodo.7293917.All high-throughput RNA sequencing data as well as transcript quantifications have been deposited at the Gene Expression Omnibus under accession number GSE194271. Genome browser views for Cyp2c70 (gkac1136_Supplemental_FilesClick here for additional data file.


In this announcement, we present the set of putative terpene synthase (TS) gene fragments detected in a subseafloor sediment sample collected off Shimokita Peninsula, Japan. This data set contains sequences with 72 to 100% identity to TS from actinobacteria and cyanobacteria. Terpenoids (TRPs) are the largest class of specialized metabolites , and manSubseafloor sediments represent an environment with unique microbiological communities and metabolic activities . HoweverChikyu shakedown cruise of CK-06-06  and frozen at −80°C immediately after the sampling. DNA was extracted from 5 g of the frozen sediment as previously described (The sediment sample was collected during the D/V TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATCGAGATGCGSCGCAAGG-3′) and geosmin-rev (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGASCGSAKGTGCCACTCGTG-3′) primers . The 2-MIB TS primers were mib-for (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACGACDNBTACTGCGAGGAC-3′) and mib-rev (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGVCGGAAGTTGTTGAACTG-3′) (331 bp). The PCR mix consisted of 1× EmeraldAmp Max PCR master mix (TaKaRa), 0.4 μM each primer, and 0.05 ng of sediment DNA. The two-phase touchdown PCR protocol for increased specificity and sensitivity was used (The geosmin TS fragment (432 bp) was amplified using primers geosmin-for . OTUs were filtered for chimeras using VSEARCH’s implementation of UCHIME de novo (The obtained sequences  were pro de novo  training de novo .This data set can be used in studies on TS gene diversity and distribution in subseafloor environments.PRJNA846928. The GenBank accession numbers for OTUs are ON723903 to ON723912 (2-MIB) and ON723913 to ON723935 (geosmin).Raw reads were deposited in a BioProject at DDBJ/ENA/GenBank under the accession number


In the published article, there were errors. In the original article, there was an error in the description of the Reverse primer sequence in Materials and methods.Materials and methods, “Deep amplicon sequencing of CMV-UL40 genomic DNA”.A correction has been made to This sentence previously stated:“The following forward and reverse primers were used: Forward, 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACAGTCGGCAGAATGAAC-3′ and Reverse, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGAACACGACGCATA-3’.”The corrected sentence appears below:“The following forward and reverse primers were used: Forward, 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACAGTCGGCAGAATGAAC-3′ and Reverse, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGAACACGAGCGGACATA-3’.”Materials and methods.In the original article, there was another error in the description of the concentration of the anti-HLA-E antibody in Materials and methods, “Immunohistochemistry analysis”.A correction has been made to This sentence previously stated:“After antigen retrieval with boiling citrate buffer (pH 6.0), the sections were incubated with 5% skim milk for 1 h at room temperature to prevent nonspecific binding and stained with 10 μL/mL anti-HLA-E antibody [MEM-E/02]  or IgG from mouse serum (Sigma-Aldrich) overnight at 4°C.”The corrected sentence appears below:“After antigen retrieval with boiling citrate buffer (pH 6.0), the sections were incubated with 5% skim milk for 1 h at room temperature to prevent nonspecific binding and stained with 10 μg/mL anti-HLA-E antibody [MEM-E/02]  or IgG from mouse serum (Sigma-Aldrich) overnight at 4°C.”The authors apologize for these errors and state that these do not change the scientific conclusions of the article in any way. The original article has been updated.


We report an approach of combining CRISPR-Cas9 gene editing with florescence cell sorting to tag and purify human endogenous protein complexes from HEK cells for structural studies by single-particle cryogenic electron microscopy. This procedure is demonstrated by applying the method to study human proteasomal complexes, enabling rapid determination of high-resolution structures of several proteasomal complexes. We envision this approach will enable structural, biochemical, and biophysical studies of many important endogenous human macromolecular complexes. The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ–20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources. Escherichia coli), insect (Sf9), and mammalian (HEK) cells. Overexpression approaches frequently encounter issues with proteins that are misfolded, nonfunctional, or degraded by the host cells. Overexpression of certain proteins may also alter protein homeostasis within cells. Substantial optimization is often necessary to identify the right conditions to produce suitable proteins for study, which is time-consuming and challenging in many cases. The problem is compounded further by multiprotein complexes where multiple subunits need to be assembled in the correct order and stoichiometries, making the coexpression of multiple subunits and purification of protein assemblies even more challenging.Recent technological breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have greatly accelerated the pace of high-resolution structure determination of biological macromolecules. However, a major bottleneck in the structural study of important targets including membrane proteins, protein–DNA complexes, and large protein assemblies is sample production. The challenge of producing functional proteins extends beyond structural biology and impacts many areas of biological sciences that currently rely on techniques of protein overexpression. Conventional methods of protein production involve overexpression of a target gene from a plasmid within heterologous systems, including bacterial . Selecting the β4-StrepII cell population, which had the highest knock-in efficiency, we expanded this cell line and purified the human proteasome by StrepII pull-down.To extract the endogenous human proteasome for structural studies, we incorporated a mNG2[11]-StrepII tag onto the C terminus of the 20S β1 (Psmb6), β2 (Psmb7), and β4 (Psmb2) B subunitSI Appendix, Fig. S2), followed by cryo-EM structure determination of singly capped proteasomal complexes of PA28–20S, PA200–20S, and 19S–20S at ∼2.6 to ∼3.2 Å resolution, with and without MG-132 inhibitor .Affinity purification of human endogenous 20S(β4-StrepII) pulls down multiple proteasomal complexes, including 20S, singly capped with 19S, PA200, and PA28. The purified proteasomal complexes were first visualized by negative-stain EM . Furthermore, the resulting cryo-EM map of PA28–20S purified from 20S(β4-StrepII) pull-down shows side-chain density that matches PA28β in some regions while matching PA28γ in other regions , suggesting that the cryo-EM map is an average of both PA28αβ and PA28γ complexes. However, the high similarity between the two complexes prevented using computational classification to separate them (Endogenous PA28 subunits are known to form two different heptameric complexes: a heteroheptamer composed of PA28α and PA28β subunits and a homoheptamer composed of PA28γ subunits. Pull-down of 20S(β4-StrepII) is expected to yield both PA28αβ–20S and PA28γ–20S complexes. Indeed, mass spectrometry analysis of the purified proteins reveal all three PA28 subunits to be present in the purified sample . Additionally, the tagging of PA28α with sfCherry2[11]-ALFA tag was performed in the cell line that already contained mNG2[11]-StrepII–tagged 20S β4, demonstrating the ability to multiplex orthogonal tags onto different subunits of a larger protein assembly to increase target specificity. The cryo-EM map of PA28αβ–20S purified by ALFA-PA28α pull-down allowed robust modeling of the side-chain densities of PA28αβ, allowing us to distinguish the subunit stoichiometry of the complex.To isolate a homogeneous PA28–20S complex for structural studies, we engineered an sfCherry2[11]-ALFA tag onto the N terminus of the PA28α subunit using the method described above, except here we used a split sfCherry system  rather tSI Appendix, Fig. S6 and Table S2), different from the 4α/3β assembly observed previously.Previous crystallographic structural analysis of the mouse PA28αβ heteroheptamer found a stoichiometry of four α and three β subunits . The proSI Appendix, Fig. S9). There is one low-occupancy binding site situated between the 20S α1 and α2 subunits where a phenylalanine, 20S α1 F29, may be sterically hindering the binding of the C-terminal tail of PA28β . We suspect that this additional density does not represent endogenous substrates but rather is from ALFA peptide used to elute purified complex. ALFA peptide is present at high concentrations in the elusion buffer and likely enters 20S as substrates through the α-ring gate opened by PA28. Consistent with this argument, cryo-EM analysis of proteasomal complexes purified via StrepII pull-down of 20S(β4-StrepII) did not show similar density inside any map of 20S, PA200–20S, or 19S–20S.Proteasome activators serve to open the 20S gate and facilitate the loading of unfolded polypeptides into the 20S inner chambers for degradation. Passing through the open gate in the α-ring, unfolded polypeptides first enter the antechamber formed between an α- and β-ring and then move into the proteolytic chamber formed between two β-rings for peptide cleavage. Interestingly, the cryo-EM map of PA28αβ–20S purified by ALFA-PA28α pull-down shows additional density inside the 20S antechamber that is proximal to the bound PA28 , which may serve to bind unfolded polypeptides. The ALFA peptide accumulates in the 20S antechamber and is likely degraded once it enters the proteolytic chamber. To visualize what might happen if proteolysis was blocked, we purified proteasome complexes via 20S(β4-StrepII) by StrepII pull-down in the presence of the peptide-like inhibitor MG-132 and carried out cryo-EM analysis , consistent with a previous study of the yeast proteasome , and signal amplification using our recent tag-assisted split enzyme complementation method  could fuOne exciting future direction stemming from the concepts described here is the study of endogenous proteins from animal models. This can allow the structural and biophysical characterization of proteins in their physiological states, which can provide important insights into how proteins function in vivo . EfficieTAATACGACTCACTATAGAAAAAAAGCACCGACTCGGTGCAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAACGATGTTAGGAGCCCTGTTTGGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGTTATTGCATACTAGAATCCCGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGGCCACCCACTGATGCCATTCGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGCCCGGTCATGGCCATGCTCAGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTAACATCATGTCCTCCCTCCCACTTGCCAGGGAACTTTTTTTTGATGGGCTCCTTTATTGACAAAAATGGCATCCATGACCTGGATAACATTTCCTTCCCCAAACAGGGCTCCGGCAGCACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTGAATACTGGGATTCTAGTATGCAATAAGAGATGCCCTGTACTGATGCAAAATTTACAAGTACTTTTGGGAGACCAGATACCCAAATTCGCCGTTGCCACTTTACCACCCGCCGGCAGCACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTGAATGGCATCAGTGGGTGGCTGGCCGCGGTTCTGGAAGGTGGTGAGCATTGAGGCATCACTCCTCTGGAGATTGAGGTGCTGGAAGAAACAGTCCAAACAATGGACACTTCCGGCAGCCCTTCTAGATTAGAAGAGGAATTGAGACGACGATTGACTGAACCAGGATCTTATACTATCGTTGAACAATACGAGAGAGCTGAGGCAAGACACTCTACAGGTTCTGGCGCCATGCTCAGGGTCCAGCCCGAGGCCCAAGCCAAGGTGAGCGCCGCCCACTCCACTCCTTGTGCGGCGCTAGGCCCCCCGTCCCGGTCATGSI Appendix, Fig. S1). The reactions were completed in a 100-μL reaction containing 50 μL 2× Phusion master mix, 2 μL ML557 + 558 mix at 50 μM, 0.5 μL ML611 at 4 μM, 0.5 μL of each gene-specific oligo at 4 μM, and 47 μL diethyl pyrocarbonate (DEPC) H2O. PCR program: 95 °C, 3 min; 20× ; 72 °C, 60 s. The PCR product was purified using a Zymo DNA Clean and Concentrator Kit (Zymo Research D4013) and DNA was eluted in 10 μL. The expected DNA concentration is around 100 ng/µL. DNA was stored at −20 °C until used for gRNA synthesis.The IVT template for LMNA gRNA was made by PCR (In vitro transcription was carried out using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB E2050S) with the addition of RNAsin (Promega N2111). In 20 μL, 300 ng of DNA template, 10 μL NTP buffer mix, 2 μL T7 polymerase, 1 μL RNAsin, and water was added. Addition of RNAsin is important to prevent RNA degradation. The reaction was incubated overnight and the RNA was purified using the RNA Clean and Concentrator Kit (Zymo Research R1017). RNA was eluted in 10 μL with an expected concentration >6 μg/μL. Single-guide RNA (sgRNA) was stored at −80 °C immediately after measuring concentration and diluting to 130 μM.2O, and 4 μL sgRNA were mixed and incubated at 70 °C for 5 min to refold the gRNA. During this step, 10-μL aliquots of purified Cas9 at 40 μM were thawed on ice and once thawed were slowly added to the diluted sgRNA in Cas9 buffer and incubated at 37 °C for 10 min for ribonucleoprotein complex formation. Six microliters of HDR template donor was added to the ribonucleoprotein mix, and all samples were kept on ice until ready for nucleofection. For efficient recovery post-knock-in (KI), a six-well plate with 2 mL of Expi293F expression media (Thermo Fisher) per well was incubated at 37 °C. An appropriate amount of supplemented Lonza Amaxa solution corresponding to the number of KIs to be performed was prepared at room temperature in the cell culture hood. For each sample, 65.6 μL of SF solution (Lonza Group Ltd.) and 14.4 μL of supplement was added to an Eppendorf tube for a total of 80 μL per KI. Lonza nucleofector instruments/computers were then turned on and kept ready for nucleofection. The Amaxa solution is toxic to cells, so it is important to make sure everything is ready to go beforehand.In a sterile PCR or microcentrifuge tube, 8 μL Cas9 Buffer, 12 μL DEPC Hg for 3 min.Supernatant was removed and cells were resuspended in 1 mL of phosphate-buffered saline to wash. The cells were centrifuged again at 500 × g for 3 min. PCR tubes containing ribonucleoproteins were brought into the tissue culture hood. Cells were resuspended in 80 μL of supplemented Amaxa solution and the cell resuspension was added to the 40 μL of preformed ribonucleoprotein + HDR template mix. The 120-μL mixture was pipetted into the bottom of the nucleofection plate, avoiding air bubbles. The nucleofection was carried out in 100-μL cuvettes on a Lonza Nucleofector X Unit (Lonza AAF-1002X) attached to Lonza 4D Nucleofector Core Unit (Lonza AAF-1002B). Cells were nucleofected using the FS-100 program and immediately recovered using 400 μL of media from the prewarmed six-well plate and transferred to the corresponding well. Because the Amaxa solution is toxic to the cells, it is important to perform these steps quickly. The cells were incubated at 37 °C and 8% CO2.Cells  were harvested by centrifugation at 500 × 2. The next day, the cells were transferred to a 125-mL vented flat-bottom Erlenmeyer flask containing 20 mL Expi293F media and incubated at 37 °C and 8% CO2 with shaking at 120 rpm until the cell density reached 1 million/mL.Cells were monitored over 5 to 7 d. Many cells will look unhealthy and die, and growth rate will slow dramatically. Cells will begin to recover after 5 d. Once cells expanded to the point where they began to detach from the well bottom, the plate was transferred to a shaker operating at 120 rpm and incubated at 37 °C and 8% COTo prepare the transfection mixture, 20 μg of pSFFV-mNG2 (1-10) plasmid was added to 1 mL of Opti-MEM (Gibco) in one tube and 53 μL of ExpiFectamine 293 (Gibco) was added to 1 mL of Opti-MEM (Gibco) in a separate tube. The mixture was incubated at room temperature for 5 min. The Opti-MEM containing Expifectamine 293 was then added to the solution containing DNA, inverted multiple times to mix, and then incubated at room temperature for 25 min. The transfection mixture was then added to 20 million Expi293F cells in 20 mL of media in a 125-mL vented flat-bottom Erlenmeyer flask. The cells were incubated at 37 °C for 2 d with shaking at 120 rpm. If the knock-in was successful, some cells will show green fluorescence the next day, which will get brighter on day 2.2 for 2 d with shaking at 120 rpm. Cells were transferred to 20 mL of Expi293F media in a 125-mL vented Erlenmeyer flask. When the cell density reached 2 × 106 per mL, cells were transferred to 100 mL of Expi293F media in a 500-mL vented Erlenmeyer flask. When cells reached a density of 2 × 106 per mL, cells were harvested and frozen in aliquots.FACS sorting and flow cytometry were performed on a BD FACSAria II in the Laboratory for Cell Analysis at the University of California, San Francisco (UCSF). mNG2 signal was measured with the 488-nm laser and 530/30 bandpass filter. One million cells per sample were sorted into Eppendorf tubes and then plated in a six-well plate. The six-well plate was incubated at 37 °C and 8% COTotal RNA was extracted from 1 million cells using the Monarch Total RNA Miniprep Kit (NEB T2010S). cDNA was prepared from 1 µg of extracted RNA using LunaScript RT SuperMix Kit (NEB E3010). No Template and No Reverse Transcriptase controls (NTC and NRT) were performed in parallel to cDNA preparations. cDNA was analyzed in a 2% agarose gel. Sequencing confirmation of amplicons was completed by Elimbio.2, and shaking at 120 rpm. When cells reached a density of 3 million cells per mL, cells were harvested by centrifugation at 1,000 × g for 5 min. Cells were resuspended in 20 mL Buffer A  supplemented with SigmaFast protease inhibitor (Sigma). Cells were lysed by sonication and insoluble debris were removed by ultracentrifugation at 100,000 × g for 20 min. For StrepII-tagged proteins, the supernatant was passed through a column containing 0.2 mL Strep-Tactin XT Sepharose resin (Cytiva) by gravity flow. The resin was washed with 2 mL Buffer A and the bound proteins were eluted with 1 mL Buffer A supplemented with 50 mM biotin . For ALFA-tagged proteins, the supernatant was passed through a column containing 0.2 mL ALFA Selector PE resin (NanoTag Biotechnologies) by gravity flow. The resin was washed with 2 mL Buffer A and the bound proteins were eluted at room temperature with 1 mL Buffer A supplemented with 200 μM ALFA peptide . To acquire 19S-bound proteasome complexes, 1 mM AMPPNP (Sigma) and 2 mM MgCl were included throughout the purification. To acquire MG-132 bound proteasomal complexes, cells were treated with 5 μM MG-132 for 15 min prior to harvesting and MG-132 was maintained throughout the purification, along with 1 mM ATP. The eluted proteins were concentrated in a 100 K centrifugal concentrating device.Expi293F cell lines were grown in 100 mL Expi293 expression medium (Thermo Fisher) in 500-mL flat-bottom Erlenmeyer flasks at 37 °C, 8% CONegative-stain EM grids were prepared following established protocol . SpecificisTEM . For PSMB2-mNG2 (11)-strepII with MG132, the 19S-20S classes were grouped and subjected to another round of 3D classification using cryoSPARC multirefinement (20-Å resolution limit) with eight classes to separate the different conformations. The resulting classes were refined to high resolution using cryoSPARC homogeneous refinement . Maps were filtered by local resolution in cryoSPARC. Atomic models were built into the cryo-EM density maps using Coot -strepII and mCherry -strepII . Images cisTEM . Particling Coot , 39 and ing Coot , 41 withing Coot .Supplementary File