Cellular totipotency is critical for whole-organism generation, yet how totipotency is established remains poorly illustrated. Abundant transposable elements (TEs) are activated in totipotent cells, which is critical for embryonic totipotency. Here, we show that the histone chaperone RBBP4, but not its homolog RBBP7, is indispensable for maintaining the identity of mouse embryonic stem cells (mESCs). Auxin-induced degradation of RBBP4, but not RBBP7, reprograms mESCs to the totipotent 2C-like cells. Also, loss of RBBP4 enhances transition from mESCs to trophoblast cells. Mechanistically, RBBP4 binds to the endogenous retroviruses (ERVs) and functions as an upstream regulator by recruiting G9a to deposit H3K9me2 on ERVL elements, and recruiting KAP1 to deposit H3K9me3 on ERV1/ERVK elements, respectively. Moreover, RBBP4 facilitates the maintenance of nucleosome occupancy at the ERVK and ERVL sites within heterochromatin regions through the chromatin remodeler CHD4. RBBP4 depletion leads to the loss of the heterochromatin marks and activation of TEs and 2C genes. Together, our findings illustrate that RBBP4 is required for heterochromatin assembly and is a critical barrier for inducing cell fate transition from pluripotency to totipotency. Cdx2, Elf5 and Eomes) could lead to the direct conversion of mESCs into TrE-like cells  blastomeres have the capacity to develop into all embryonic and extraembryonic cell types, exhibiting a totipotent state ,2. The tke cells , but linke cells ,26. TherDux, Zscan4 and Nelfa) have been found to facilitate the generation of 2CLCs from mESCs  has been identified among cultured mESCs, and several TF genes  and 2C genes, and enhances trophoblast formation. Remarkably, RBBP4 is co-enriched at all three classes of ERVs  with either H3K9me2 or H3K9me3. Intriguingly, RBBP4 could simultaneously recruit G9a and KAP1 to deposit H3K9me2 and H3K9me3 on the TEs, respectively. Moreover, RBBP4 increases nucleosome density at TE sites, and RBBP4 depletion reduces chromatin binding of both H3K9me2 and H3K9me3, and reprograms the pluripotent stem cells toward 2C-like totipotent cells.2 at 37°C. RBBP4 or RBBP7 degradation was induced by treating mESCs with a final concentration of 500 μM indole-3-acetic acid .The 46C mESCs were cultured on 0.1% gelatin-coated plates with the mESCs medium containing high-glucose Dulbecco's modified Eagle's medium , 15% fetal bovine serum , 1× GlutaMAX , 1× non-essential amino acids , 1× sodium pyruvate , 0.1 mM 2-mercaptoethanol , 3 μM Chir99021 , 1 μM PD0325901  and 1000 U/ml leukemia inhibitory factor (LIF). HEK293T cells  were maintained in high-glucose DMEM supplemented with 10% FBS . All cell lines were cultured under 5% CORbbp4 or Rbbp7 donor plasmid, a DNA fragment containing the stop site (∼1 kb), which was amplified by polymerase chain reaction (PCR) from the genomic DNA of 46C mESCs, was cloned into a pEASY-Blunt plasmid . After creating a restriction enzyme BamHI site by inverse PCR, a DNA fragment containing the AID–mCherry tag linked to a SV40 promoter-driven neomycin/kanamycin resistance gene (AID–mCherry-NeoR) was cloned into the donor plasmid. The AID–mCherry-NeoR backbone was cut from the PTJ58 vector by BamHI. The specific guide RNA (gRNA) targeting the 3′-end sequences of the mouse Rbbp4 or Rbbp7 gene were designed and inserted into a pX330 plasmid , respectively. The single guide RNA (sgRNA) oligos are available in We used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system to construct the gene-edited cell lines. To construct the OsTIR1 were established by co-transfection of the plasmids pEN396  and pX330-EN1201  expressing a gRNA targeting the Tigre locus using FuGENE6 Transfection Reagent  following the manufacturer's instructions. Clones were selected with 1 μg/ml puromycin for 3 days. After validation of the OsTIR1 expression cassette integration into the Tiger locus, the donor plasmids and their corresponding sgRNA targeting Rbbp4 or Rbbp7 were co-transfected into the parental cell lines. The transfected cells were selected with 200 μg/ml G418 for 5 days. Colonies were manually picked up and were further validated by PCR for homozygous insertion of the sequences encoding AID–mCherry.The parental mESCs expressing an Rbbp4 and Rbbp7 cDNAs were cloned into the pSin-Flag-Avitag vector with a puromycin resistance gene. For Rbbp4 knockdown, the shRNA oligos targeting Rbbp4 were inserted into the lentiviral vector pLKO.1. All sequences of the plasmids were validated by Sanger sequencing before using for further experiments. For lentivirus generation, HEK293T cells were plated, cultured overnight and then co-transfected with pSin or pLKO.1 vector containing target genes together with the packaging plasmids psPAX2 and pMD2.G by using polyethyleneimine . The culture medium was changed after 12 h. The viral supernatants were collected 48 h post-transfection. After filtration through a 0.45 μm filter, the supernatants were used to infect mESCs. The sequences of shRNA oligos used in this study are listed in For overexpression, Rbbp4 and pSin-Flag-Avitag-Rbbp7 lentivirus, respectively. Thereafter, the cells were selected with 2 μg/ml puromycin for 3 days. Finally, in vivo biotinylation of RBBP4 and RBBP7 was detected with anti-Biotin antibody .The 46C mESCs were infected with lenti-birAV5 lentivirus and selected with 5 μg/ml blasticidin for 4 days. Then BirAV5-overexpressing cells were infected with pSin-FLAG-Avitag, pSin-Flag-Avitag-Cdx2-tdTomato mESCs were trypsinized and then resuspended in trophoblast stem cell (TSC) medium containing RPMI1640 , 20% FBS, 1× GlutaMAX, 1× sodium pyruvate, 1× penicillin–streptomycin, 0.1 mM 2-mercaptoethanol, 2 ng/ml transforming growth factor-β1 , 25 ng/ml fibroblast growth factor 4  and 1 μg/ml heparin . Then cells were replated at a density of 1000–5000 cells per well in 12-well plates and cultured in TSC medium. The culture medium was changed daily.Cell culture plates were pre-coated with 0.3% gelatin. The cells were lysed in RIPA lysis buffer  on ice for 30 min and the supernatants were obtained by centrifuging at 12000 rpm at 4°C for 10 min. The boiled proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinyldifluoridene (PVDF) membranes. The membranes were incubated with primary antibodies at room temperature (RT) for 2 h or at 4°C overnight after blocking with 5% milk. The membranes were washed with phosphate-buffered saline (PBS) containing 0.1% Tween-20 and then incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) at RT for 1 h. ECL solution was added and the signals were detected using a MiniChemi™ Chemiluminescence Imaging System (Beijing Sage Creation). The primary antibodies used herein are as follows: RBBP4 , RBBP7 , KAP1 , CHD4 , G9a , HP1α , β-Actin  and glyceraldehyde phosphate dehydrogenase .Approximately 5000 cells were plated per well of 96-well plates. The next day, the cells were cultured in 100 μl of mES culture medium with or without IAA (this time point was recorded as day 0). The cell viability was detected at the indicated time points by using Cell Counting Kit-8  according to the manufacturer's instructions. In brief, 10 μl of CCK8 solution was added to each well and incubated for 1.5 h, and the absorbance at 450 nm was measured using a spectrophotometer.Apoptosis assays were performed using the Annexin V-FITC Apoptosis Detection Kit  according to the manufacturer's instructions. In brief, 30000 cells were plated per well of 6-well plates and treated with 500 μM IAA or PBS for 24 h before collection. The supernatants of the culture medium were collected. The adherent cells were washed once with PBS, digested with trypsin without EDTA and collected together with the supernatants. The cells were centrifuged and washed once with PBS. The cells were resuspended in 100 μl of 1× Annexin V binding buffer. Then 5 μl of Annexin V–fluorescein isothiocyanate (FITC) was added to the cell suspension and incubated in the dark at RT for 10 min. Subsequently, 400 μl of 1× Annexin V binding buffer was added to each sample and the fluorescence intensity was detected using an LSRFortessa flow cytometer (BD Biosciences) within 1 h.A total of 40000 cells were plated per well of 6-well plates and treated with IAA for 24 h before collection. The cells were washed and then fixed with pre-cooled 75% ethanol overnight at 4°C. The cells were washed once with PBS, resuspended with 100 μl of PBS containing 0.25 μg of 4′,6-diamidino-2-phenylindole  and incubated in the dark at RT for 15 min. Finally, 500 μl of PBS was added to each sample after washing and the fluorescence intensity was detected using an LSRFortessa flow cytometer (BD Biosciences).AP staining was performed following the protocol of the BCIP/NBT Alkaline Phosphatase Color Development Kit . In brief, the cells were fixed with 4% paraformaldehyde (PFA) solution for 5 min and gently washed, followed by staining with BCIP/NBT solution in the dark at RT for 30 min.Rbbp4-overexpressing 46C mESCs were collected and lysed with cell lysis buffer . The supernatants were obtained by centrifugation at 12000 rpm at 4°C for 10 min and 1 mg of proteins were used for immunoprecipitation. A 15 μl aliquot of M280 Streptavidin Dynabeads  was added to the samples and incubated overnight at 4°C with gentle rotation. Beads were washed four times using 1 ml of cell lysis buffer at 4°C for 10 min each time. Immunoprecipitated proteins were visualized by western blots.Flag-Avitag- or Flag-Avitag-® III RT SuperMix for quantitative PCR . Quantitative reverse transciption–PCR (RT–qPCR) was performed using SYBR Green mix  on the CFX96 system (Bio-Rad). Gene expression levels were normalized to Gapdh. The primers used in the RT–qPCR assays are listed in To extract the total RNAs, the cells were lysed with TRIzol reagent , followed by addition of 1/5 volume of chloroform. After centrifugation at 12000 rpm at 4°C for 10 min, the total RNAs in the supernatants were precipitated with isopropanol. cDNAs were synthesized with HiScriptCells were fixed with 4% PFA at RT for 10 min, permeabilized with 0.5% Triton X-100 at RT for 10 min and blocked with 3% bovine serum albumin (BSA) at RT for 1 h. Samples were then incubated with primary antibody overnight at 4°C and subsequently with secondary antibody. A final concentration of 1 μg/ml DAPI  was used to stain nuclei. Fluorescence images were captured with a ZEISS Axio Observer 7. The EOMES antibody  used in this study was diluted 1:500.The total RNAs were extracted as described above. RNA sequencing libraries were constructed by using a VAHTS mRNA-seq V3 Library Prep Kit  according to the manufacturer's instructions. In brief, mRNA capture beads were used to capture poly(A)-enriched RNAs from 1.5 μg of total RNAs and then the RNAs were fragmented at 85°C for 6 min. Next, the first-strand and second-strand cDNAs were synthesized. The cDNAs were purified with AMPure XP beads , followed by end repair, adaptor-ligation, size selection and library amplification. Finally, the libraries were purified using AMPure XP beads and then sequenced on a NovaSeq 6000 sequencer.scRNA-seq libraries were prepared by using a DNBelab C Series Single-Cell Library Prep set . In brief, the dissociated cell suspensions were converted to barcoded scRNA-seq libraries through droplet encapsulation, emulsion breakage, mRNA captured bead collection, reverse transcription, cDNA amplification and purification. The libraries were sequenced on a DNBSEQ-T7 sequencer.5 cells were washed twice with wash buffer  by gentle pipetting. A 10 μl aliquot of concanavalin A-coated magnetic beads  was activated and added per sample and incubated at RT for 10 min. The supernatants were removed and bead-bound cells were resuspended in 100 μl of wash buffer containing 0.01% digitonin and 2 mM EDTA. The H3K9me2 antibody  was added and incubated overnight at 4°C on a rotator. The beads were washed and resuspended in 300-wash buffer containing 300 mM NaCl with pG-Tn5  at RT for 1 h. The beads were washed and tagmentation was performed in 300-wash buffer supplemented with 10 mM MgCl2 at 37°C for 1 h. To stop the tagmentation reaction, 2.25 μl of 0.5 M EDTA, 2.75 μl of 10% SDS and 5 μl of proteinase K (20 mg/ml) were added and further incubated at 55°C for 2 h. Then the genomic DNAs were extracted by phenol–chloroform and subjected to PCR amplification using NEBNext High-Fidelity 2× PCR Master Mix  for 13 cycles. The libraries were size-selected with 1.2× AMpure XP beads  and sequenced on a NovaSeq 6000 sequencer.CUT&Tag assays were performed as described previously with some modifications . Briefly5 cells were harvested, washed and bound to activated concanavalin A-coated magnetic beads as for CUT&Tag. The bead-bound cells were incubated with primary antibody in 100 μl containing 0.01% digitonin wash buffer  on a rotator at 4°C overnight. The primary antibodies used herein are as follows: H3K9me3 , KAP1 , CHD4  and G9a . After two washes, the beads were resuspended in 100 μl of wash buffer containing pAG-MNase and incubated at 4°C for 1 h. After two washes in wash buffer and one wash in low-salt rinse buffer , the tubes were chilled to 0°C and the beads were resuspended in 100 μl of ice-cold calcium incubation buffer . The tubes were kept on ice 30 min, followed by immediate addition of STOP buffer . The beads were incubated at 37°C for 30 min and the liquid was removed to a fresh tube on a magnet stand. Then SDS and proteinase K were added and incubated at 55°C for 2 h. The genomic DNAs were extracted by phenol–chloroform and the sequencing libraries were prepared through VAHTS Universal DNA Library Prep Kit , and sequenced on a NovaSeq 6000 sequencer.CUT&RUN assays were performed as described previously with some modifications . Briefly6 cells were harvested, washed in ice-cold PBS and resuspended in 1 ml of ice-cold lysis buffer  and rotated at 4°C for 15 min. Nuclei were then pelleted at 300 g at 4°C for 10 min and washed in 1 ml of digestion buffer . MNase digestion was carried out in 100 μl of digestion buffer containing 2 mM CaCl2 and 2500 gel units of MNase  with shaking at 37°C for 5 min. The reaction was stopped using an equal volume of stop buffer  before RNase A and proteinase K treatment. The gDNAs were extracted by phenol–chloroform. Purified DNAs were loaded on a 1.5% agarose gel to determine digestion efficiency. The mononucleosome–DNAs were purified by AMPure XP beads . The sequencing libraries were prepared from 10 ng of purified mononucleosome–DNAs using the VAHTS® Universal DNA Library Prep Kit  with 10 cycles of PCR, and sequenced on a NovaSeq 6000 sequencer.MNase-seq was performed as described previously with some modifications ,53. In bBiotin ChIP-seq was performed as described . In brieP <0.01 and an absolute fold change (FC) >2 (for genes) or an absolute FC >1.5 (for TEs).To analyze the expression of genes and TEs, RNA-seq raw reads were trimmed using Trim_galore (v0.6.7) and aligned to the mouse genome (mm10 and Gencode gene annotation vM25) using STAR (v2.7.0d)  and RSEMFor principal component analysis (PCA) , the aliRaw reads were trimmed using Trim_galore (v0.6.7) and mapped to the mouse genome (mm10) using Bowtie2 (v2.2.5) . For TE P <0.05 and an absolute FC >1.5.RBBP4, RBBP7, G9a and KAP1 peaks were called by MACS2 (v2.2.7.1)  with the2(fold enrichment). Heatmaps and pile ups were generated using DeepTools (v3.5.1).For analysis of TEs, repeat element annotations (RepeatMasker) were downloaded from the UCSC genome browser database (mm10). TEs shorter than 300 bp or those TEs with <50 copies were removed from the analysis. RBBP4 enrichment on TEs was calculated by dividing the observed value by the input and was expressed as the logRaw reads were trimmed by Trim_galore (v0.6.7) and mapped to the mouse genome (mm10) using Bowtie2. DANPOS3  was usedNucleosome-free regions were evaluated as previously described with some modifications . RegionsSmart-Seq2 read files were mapped to the mouse genome (mm10 and Gencode gene annotation vM25) using STAR (v2.7.0d)  and RSEMhttps://github.com/MGI-tech-bioinformatics/DNBelab_C_Series_HT_scRNA-analysis-software. Cells with <200 expressed genes detected or >10% of mitochondrial reads were filtered out.The analysis of DNBeLab C4 scRNA-seq data followed: Analysis of the filtered data was conducted in R (v4.2.2) using the Seurat suite (v4.1.1) . The intt-test analysis (two-tailed) for two groups, unless otherwise indicated. Differences in means were considered statistically significant at P <0.05. Significance levels were: *P <0.05; **P <0.01; and ***P <0.001.Data are presented as mean values ± standard deviaition (SD) unless otherwise indicated in the figure legends. Sample numbers and experimental repeats are indicated in the figure legends. Statistical significance was determined by Student's Rbbp4 and Rbbp7 mESC lines   and MERVL at 24 h after RBBP4 depletion  and the population of MERVL-tdTomato cells were significantly increased in the Rbbp4-depleted cells  Figure , which a) Figure ,28,74. C) Figure , we furt) Figure . RT–qPCRn Figure . Conversn Figure –G. To fun Figure  were useA oligos . Our dated cells , but noted cells .Rbbp4-AID mESCs cultured with or without IAA treatment, and integrated the data with published single-cell transcriptomes of mouse early embryos  at a high level and pluripotency genes (such as Oct4 and Nanog) at a low level  and Cdx2-tdTomato dual fluorescent reporter (OG-CT) mESCs (Rbbp4-depleted cells in mouse TSC medium (Rbbp4-depleted OG-CT mESCs activated the expression of 2C markers (MERVL and Zscan4)  was decreased in all differentiated cells  mESCs  to exploC medium   . mESCs ce Figure . We nexted cells  and the nd Ascl2 ,24,83–85s Figure , E. Flows Figure . Our rescis-regulatory elements to regulate expression of their neighboring genes  <0.01] and RBBP4-bound TEs were <1 kb  and a small percentage at promoters (14%) Figure . Intrigung genes ,87. Inteng genes . Furtherng genes . Notablyng genes . Moreoveng genes . Additiore <1 kb , suggestTo explore how RBBP4 regulates TEs, we conducted hierarchical clustering of RBBP4 peaks with both active  ,89 and rRbbp4-AID mESCs with or without IAA treatment. Our data indicated that RBBP4 degradation resulted in the significant reduction of 5068 (12%) H3K9me2 peaks (42.8% in TEs sites) and 10900 (69%) H3K9me3 peaks (81.7% in TEs sites), but caused an increase of only 612 (1.5%) H3K9me2 peaks and 50 (0.3%) H3K9me3 peaks, respectively , which recruits SETDB1 to the target sites  and with that of GLP , but was relatively weakly correlated with the expression levels of other lysine methyltransferases (R < 0.6)  . MoreoveR < 0.6) . Furtherknockout  and G9a knockout , which rknockout   of the CAF-1 complex have also been shown to regulate the establishment of heterochromatin . CHAF1A/Chd4 deficiency  can slide, eject, insert or replace histones within nucleosomes to alter chromatin structure and accessibility . In the ficiency . Moreoveficiency . Importaficiency . RBBP4 aficiency ,117. Surficiency ,119. Thi1 and S phases along with a global decrease in the G2 phase  under accession no. CRA007021. The previously published RNA-seq sequencing data that were reanalyzed here are available in the GEO under the accession codes: GSE49669 , GSE1245gkad219_Supplemental_FilesClick here for additional data file.


Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5′ splice site (5′SS). In mammals, many introns contain weak 5′SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (nuclear RNAi-defective 2), and CCDC174 (coiled-coil domain-containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and 5′SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP-specific proteins, and they are required for the selection and effective processing of weak 5′SSs. Our results reveal that mammalian cells use noncanonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5′SS sequences in hundreds of genes, promoting proper splice site choice, and accurate pre-mRNA splicing. Accurate removal of noncoding intronic sequences during pre-mRNA splicing is a prerequisite for eukaryotic gene expression. Most introns are excised by the major spliceosome, a dynamic macromolecular assembly consisting of five small nuclear ribonucleoprotein particles  and a myriad of associated splicing factors . The hig5′ splice site (5′SS) recognition by the U1 snRNP represents the first step of spliceosome assembly. 5′SSs comprise a short motif that is recognized by a complementary sequence at the 5′ end of the U1 snRNA . HoweverDue to the limited complementarity to U1 snRNA, weak 5′SSs are not efficiently recognized by the canonical U1 snRNP consisting of the U1 snRNA, a set of seven Sm proteins forming a ring structure common to other U snRNPs, and three U1-specific proteins, U1A, U1C, and U1-70K . TherefoHere, we combined genome engineering in mouse embryonic stem cells (mESCs) with proteomics and genomics approaches to analyze the function of NRDE2 in mammalian cells. In order to reliably detect potential RNA substrates of NRDE2 with high sensitivity and at a transcriptome-wide level, we have developed a modified cross-linking immunoprecipitation coupled to next-generation sequencing method. This revealed that mouse NRDE2 together with CCDC174 bind to U1 snRNAs, which target NRDE2 to 5′SSs. At weak 5′SSs with suboptimal U1 complementarity, NRDE2 and CCDC174 are necessary for correct splice site choice and efficient pre-mRNA splicing. Thus, we reveal that mammalian cells use an alternative strategy involving direct U1 snRNA interaction with noncanonical splicing factors to select and splice introns with suboptimal 5′SSs.Nrde2 knockout cells. These cells were further edited to introduce homozygous Nrde2 mutations and to tag NRDE2 with a fluorescent protein for live-cell imaging or with a composite 3xFLAG-AviTag (3A tag) for tandem FLAG-Streptavidin purifications. The knockout and tagging approaches were used on other endogenous genes in this study , which also included fusions with the 2xHA-FKBP12F36V domain (dTAG) for rapid protein depletion by the dTAG-13 compound . The efficacy of 4OHT or dTAG-13-mediated protein depletion was verified by western blotting .Using our previously established mESC line expressing 4-hydroxytamoxifen (4OHT) inducible Cre recombinase and bacterial BirA ligase , we genecompound . We asseNrde2 resulted in growth arrest , revealing NRDE2 has an essential function in cellular growth. To understand the basis for this phenotype, we mapped the NRDE2 protein–protein interaction (PPI) network by performing tandem affinity purifications of 3A-NRDE2 under mild native conditions coupled with mass spectrometry (nTAP-MS). The NRDE2 interactome was dominated by ribosomal proteins, splicing factors, and included the complete nuclear exosome . Four conserved aspartates within this region  resembled an MTREX Arch interaction motif . NRDE2-D174R protein levels were lower compared with wild-type NRDE2 , suggesting that MTREX controls NRDE2 stability. This was further supported by reduced NRDE2 levels upon Mtrex knockout , which is in line with previously published data from human cells . Strikingly, we did not detect any PPIs for NRDE2Δ200 under these conditions . Thus, NRDE2 sustains cell growth independently of its protein interactors.In NRDE2 loss-of-function experiments, the 4OHT-induced knockout of  exosome A. The nu exosome . Reciproon motif , and a san cells . Howevernditions B, indicaSupplemental Fig. S2H), suggesting that NRDE2-D174R and MTREX still colocalize in vivo despite their reduced binding affinity. In agreement with the native pull-down, only a few mostly high-abundant proteins were detected in NRDE2Δ200 xTAP-MS along with low levels of MTREX, but with no spliceosome components present  protocol, which uses a short fixation with a low-concentration formaldehyde followed by stringent lysis and washing conditions. NRDE2 xTAP-MS binding proteins were enriched for MTREX and splicing factors C, includ present D.Supplemental Table S2). Selected Y2H interactions were validated by reciprocal co-IP. Unlike MTREX, NRDE2 interactions with U2AF2 and CCDC174 required an intact RNA component . In contrast, a transcriptional shut-off by Actinomycin D led to a substantial reduction of NS signal even for wild-type NRDE2 . Overall, these results suggest that NRDE2 is actively recruited to and associated with pre-mRNAs that undergo splicing, independently of its interaction with splicing factors.Consistent with a splicing-related activity, fluorescent light microscopy revealed that NRDE2 was strongly enriched in nuclear splicing speckles (NSs) G. NSs arSupplemental Fig. S4A).To explore the RNA-binding potential of NRDE2, we developed a Benzonase-assisted RNA cross-linking immunoprecipitation protocol coupled to high-throughput sequencing (BCLIP-seq) . Our appSupplemental Fig. S4B), which allowed us to merge the replicates into one data set for most of the subsequent analyses. More than half of the mapping reads in all variant NRDE2, MTREX, and CCDC174 libraries matched rRNA . The rRNA signal in NRDE2 and CCDC174 BCLIP-seq data could therefore represent nonspecific binding to abundant RNA. However, the EIF4A3 data indicated that rRNA was not a common contaminant of the BCLIP method . When considering all expressed 5′SSs, NRDE2 and, to a lesser extent, NRDE2-D174R were enriched at a subset of 5′SSs similar to CCDC174, whereas NRDE2Δ200 and MTREX showed very little binding around 5′SSs. EIF4A3 was enriched at most 5′SSs, as expected for an EJC factor , whereas a large group of transcripts up-regulated in Nrde2-KO cells were also up-regulated upon conditional Mtrex knockout. These were highly enriched for genes defining a subpopulation of mESCs that resemble the gene expression state of two-cell (2C) embryos .RNA-sequencing (RNA-seq) analysis of ed genes A. NRDE2Δ embryos . The expgulation , and MERSupplemental Fig. S5B). When combined with knockouts of fHC-regulating enzymes G9A and KDM1A . These results indicate that NRDE2 influences MERVL expression independently of fHC formation. NRDE2 could be regulating MERVL at a post-transcriptional level, given its link to MTREX and the nuclear exosome. However, reducing the MT2_Mm reporter to shorter variants that preserve promoter activity but ultimately lead to reporter transcripts devoid of MERVL sequences did not alleviate NRDE2 dependence . Therefore, the observed up-regulation of MERVL and 2C genes upon Nrde2 or Mtrex knockout likely represents a pleiotropic effect caused by altered RNA metabolism, rather than an escape from the specific action of NRDE2 and MTREX on these genes. This conclusion is further supported by the negligible effects of the NRDE2-D174R mutation on gene expression, including 2C genes .To test the hypothesis that NRDE2 acts with MTREX to negatively regulate MERVL expression, we created cells with an MT2_Mm-driven mNeonGreen (2C reporter) inserted in a genomic locus replacing an active MERVL . In both Nrde2-KO and Ccdc174-dTAG cells, we found hundreds of misspliced up-regulated introns that were generally enriched for NRDE2 and CCDC174 binding at their 5′SSs . However, the up-regulated introns constituted only a small fraction of all the introns defined as NRDE2 and CCDC174 target introns , for which the peak-calling analysis of the BCLIP-seq data identified a NRDE2 or CCDC174 peak at their 5′SS. The majority of the target introns with NRDE2- or CCDC174-bound 5′SSs remained unaffected. Misspliced target introns largely overlapped in the Nrde2-KO and Ccdc174-dTAG data sets, but they were distinct from target introns up-regulated in Mtrex-KO cells . Hence, ablation of NRDE2 or CCDC174 can have a direct effect on the splicing of a subset of introns they associate with, which is not observed for MTREX. Therefore, NRDE2 and CCDC174 likely function independently of MTREX in pre-mRNA splicing. We note that the number of NRDE2-bound up-regulated introns was much smaller in Nrde2Δ200 compared with Nrde2-KO cells, suggesting the amino-terminally truncated NRDE2 functions as a hypomorph .Given NRDE2 and its interactor CCDC174 bind to 5′SSs, we hypothesized that pre-mRNA splicing could be defective in mutant cell lines, manifest as globally increased intronic signals in the RNA-seq data. To exclude transcription activation effects, we analyzed down-regulated transcripts only . The majority of introns remained unchanged even after CHX treatment, prompting us to investigate the selective sensitivity of target introns to NRDE2 or CCDC174 depletion. Analysis of intron features revealed that NRDE2-bound introns up-regulated in Nrde2-KO cells possess weaker 5′SSs, while their 3′SSs and branch points are comparable with nonaffected introns .Our observation that splicing of only a minor fraction of NRDE2 and CCDC174-bound introns was affected in ytoplasm . Whereas introns E. The diNrde2-KO and Ccdc174-dTAG cells resulted in an overall decrease of the respective mRNA signal . However, the impact on mRNA levels was generally small, possibly due to the stability of the aberrantly spliced transcripts. To assess the effect on translation, we performed ribosome profiling (Ribo-seq) in wild-type and Nrde2-KO cells. Compared with RNA-seq, a larger Ribo-seq signal reduction was observed for messages whose splicing was affected by Nrde2 knockout . Thus, overall, NRDE2 and CCDC174 are required for efficient splicing of a largely overlapping set of target introns with suboptimal 5′SS sequences.The aberrant splicing of introns in knockout F. For exNrde2 knockout . The first intron of the essential cell cycle regulator gene Cdk2, which contains a weak 5′SS strongly bound by NRDE2 and CCDC174, provides a representative example of such aberrant splice site choice. Examination of our RNA-seq data uncovered a shift in Cdk2 intron 1 splicing from the annotated 5′SS to several cryptic 5′SSs in cells lacking NRDE2 or CCDC174  or CWC22 resulted in minimal cryptic 5′SS usage. Notably, all the alternative 5′SSs in the Cdk2 intron 1 that are used in the absence of NRDE2 or CCDC174 consist of suboptimal sequences. Thus, NRDE2 and CCDC174 appear to be dispensable for general splicing, but are required for appropriate splice site choice.Defective splicing can lead to intron retention, splicing from a cryptic 5′SS, or splicing to a cryptic 3′SS. Analysis of novel splicing events in up-regulated NRDE2-bound introns revealed frequent cryptic 5′SS usage upon knockout A. Likewi CCDC174 B. Splici CCDC174 B. Usage  1 and 3 C. We obsCdk2 intron 1 as a NRDE2 target, we fused Cdk2 exons 1–3 with the Renilla luciferase gene in a dual-luciferase reporter plasmid (CDK2-wt5′SS). We also generated a CDK2 reporter construct in which the sequence of the first 5′SS was optimized by a single point mutation (CDK2-strong5′SS), improving U1 snRNA complementarity . A 2-nt substitution that strengthened the weak 5′SS of intron 5 abolished the NRDE2-dependency of Tti1 splicing , consistent with the CDK2 reporters.CDK2-wt5′SS activity was insensitive to the NRDE2-D174R mutation, but was affected by the amino-terminal truncation (NRDE2Δ200) E, furthestrength F, indicaCdk2 intron harbors multiple cryptic 5′SSs, which allowed us to dissect the 5′SS choice hierarchy. We introduced several mutations into the CDK2-wt5′SS luciferase reporter: mutations that improve the strength of either the first or third cryptic 5′SS, or a mutation that inactivates the annotated 5′SS . The BCLIP-seq signal covered most of the highly structured U1 snRNA sequence, with the highest enrichment over the stem–loops II and III (SL2 and SL3) for both NRDE2 and CCDC174 . U1 SL2 and SL3 and U4/U6 stem III are in close proximity in the precatalytic spliceosomal pre-B complex , an association that is highly specific, given we did not observe this for MTREX or EIF4A3 A. The po CCDC174 D, indicaThe indiscriminate association of NRDE2 and CCDC174 with 5′SSs is explained by U1 snRNA-mediated targeting. To further test this model, we decided to perturb the structure of the Sm ring complex, which is necessary for U1 snRNA maturation and trafficking . DepletiMammalian cells must cope with the challenge of selecting and splicing 5′SSs with suboptimal U1 snRNA complementarity. Mechanistic studies on weak 5′SS splicing have thus far focused on auxiliary splicing factors, mainly from the SR-protein family, that bind exonic splicing enhancer (ESE) motifs in the vicinity of 5′SSs to support the binding of canonical U1 snRNPs . Our stu2+ ions, the resulting library might lack the single-nucleotide resolution of the UV cross-linked nucleotide as suggested previously  or both Cre-ERT2 and V5-tagged BirA (cMB063) in the 26 locus  were usel-glutamine [Gibco], 0.1 mM 2-mercaptoethanol [Sigma], 50 µg/mL penicillin, 80 µg/mL streptomycin, 1:500 MycoZap Prophylactic [Lonza], homemade LIF conditioned medium, and 2i inhibitors: 3 µM GSK-3 inhibitor XVI [Sigma] and 10 µM MEK inhibitor PD0325901 [Tocris]). To induce conditional knockout of Nrde2 and Mtrex, 4OHT  was added to the medium and replaced daily for a total of 4 and 3 d, respectively. Depletion of endogenous proteins fused to the 2xHA-FKBP12F36V domain (dTAG) was achieved by treating the cells with 0.5 µM dTAG-13 compound  for 24 h. For chemical inhibition of splicing, cells were treated with 1 µM Thailanstatin A  for 6 h. To block transcription, cells were treated with 5 µg/mL Actinomycin D  for 2 h. For translation inhibition, 100 µg/mL Cycloheximide  was added for 4 h.Unless stated otherwise, cells were grown on gelatin-coated dishes in S-L-2i medium . All TALEN and Cas9 target sequences and donor sequences for homologous recombination are listed in Supplemental Table S1. Cells grown in medium without 2i inhibitors were seeded 5 × 105 per well of a six-well plate and transfected with a mixture of 500 ng of donor DNA and 500 ng of Cas9-2A-Puro/TALEN mix with pRR-Puro reporter (We used both TALEN and Cas9 nucleases to generate cell lines with endogenous gene modifications. Endogenous reporter  in Opti-Cell growth was monitored using the ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay kit (Lonza). Triplicates of 500 cells in 0.5 mL of medium were seeded on a 24-well plate. After 24 h, the medium was removed from the wells of day 0 time point and replaced with 50 µL of PBS. Next, 10 µL of ViaLight cell lysis reagent was added and mixed by pipetting, and the plate was incubated on a rocking platform for 10 min at room temperature. An amount of 10 µL of the lysate was mixed with 10 µL of AMR reagent in a 96-well solid-bottom white assay microplate (Corning) and, after 2-min incubation at room temperature, the luminescence was measured in Mithras LB 940 microplate reader (Berthold Technologies). In the wells of later time points, the medium was replaced 24 h after seeding and every following 24 h with fresh medium with or without 0.1 µM 4OHT. All the later time points were processed for luminescence measurement as described above.7 cells) were used per each replicate sample. The dishes were placed on ice, and cells were rinsed with cold PBS and collected by scraping and spinning at 3000g for 2 min at 4°C. The pellets were washed with 0.5% BSA in PBS and then resuspended in 0.5 mL of ice-cold B100 buffer  supplemented with 1× Halt Protease Inhibitor Cocktail (HALT) (Thermo Fisher Scientific) and 100 U of Benzonase (Sigma). After 30-min shaking (500 RPM) at 12°C, lysates were diluted with 450 µL of B100 + HALT and centrifuged at 16,000g for 5 min at 4°C.Each nTAP-MS was performed in three independent replicates. Three near-confluent 10-cm dishes  equivalent to 20 µL of the original slurry were resuspended in PBS with 0.02% Tween-20 and coupled with 2 µL of anti-FLAG M2 antibody (Sigma), rotating for 30 min at room temperature. Coupled beads were washed once with PBS + 0.02% Tween-20 and twice with B100. The beads were then resuspended in 50 µL of B100 + HALT, mixed with the cleared lysates, and rotated for 2 h at 4°C. Following three washes with 1 mL of ice-cold B100, FLAG-tagged proteins were eluted by 15-min shaking (500 RPM) in 25 µL of B100 + HALT containing 1 mg/mL 3xFLAG peptide (Sigma) at room temperature. The beads were further rinsed with 425 µL of B100 + HALT, and the supernatant was combined with the 25 µL of eluate from the previous step.M-280 Streptavidin Dynabeads (Thermo Fisher Scientific) equivalent to 20 µL of the original slurry were prewashed twice with B100, resuspended in 50 µL of B100 + HALT, mixed with the FLAG beads eluates, and rotated for 30 min at room temperature. The beads were then washed three times with 1 mL of B100, resuspended in 0.2 mL of TN buffer , and transferred to a new tube. While remaining immobilized on the magnetic stand, the beads were rinsed with an additional 1 mL of TN buffer.On-bead digestion for mass spectrometry was performed as follows. The washed detergent-free beads were resuspended by vortexing in 5 µL of digestion buffer  and sonicated in Bioruptor Plus (Diagenode) at high energy in 30 cycles, 10 sec ON/10 sec OFF. Next, 1 µL of 0.2 mg/mL LysC protease (Promega) in 50 mM HEPES (pH 8.5) was added, and proteins were predigested for 2 h rotating at room temperature. The urea concentration was then diluted with 17 µL of 50 mM HEPES (pH 8.5) and, after adding 1 µL of 0.2 mg/mL trypsin (Promega) in 0.2 mM HCl, the digestion continued overnight at 37°C with interval mixing at 2000 RPM for 30 sec every 15 min. Digested proteins were then subjected to mass spectrometry as described below in “Mass spectrometry analysis.”7 cells were collected by centrifugation at 300g for 2 min and 4°C. The pellets were resuspended in 1.5 mL of room temperature PBS and cross-linking started by the addition of 1.5 mL of 0.2% formaldehyde in PBS. After 10 min rotating at room temperature, 150 µL of 2.5 M glycine was added and samples were placed for 2 min on ice to quench the excess formaldehyde. Cells were pelleted, rinsed with 0.2% BSA in cold PBS, and finally resuspended by vortexing in 180 µL of TMS buffer  prechilled at 12°C and supplemented with 100 U of Benzonase. Following 30-min incubation at 12°C, the lysates were diluted with 1.57 mL of DIL. mix containing a 4:5 (v/v) ratio of H2O and dilution buffer , supplemented with 1× HALT and incubated for 5 min on ice. Any remaining insoluble material was removed by spinning at 16,000g for 5 min at 4°C.Each xTAP-MS was performed in three independent replicates. For each replicate sample, cells were harvested by trypsinization, placed on ice, and counted on a Countess II Automated Cell Counter (Thermo Fisher Scientific), and 4.5 × 10The cleared lysates were mixed with anti-FLAG antibody-coupled Protein G Dynabeads, which were prepared essentially as described above in the nTAP-MS section, but prewashed with 2 × 1 mL of WASH buffer  and resuspended in 50 µL of DIL. mix. After 2 h rotating at 4°C, the beads were washed three times with 1 mL of cold WASH buffer and FLAG-tagged proteins were eluted by shaking at 500 RPM for 15 min in 25 µL of TEDS buffer  containing 1 mg/mL 3xFLAG peptide at room temperature. The beads were rinsed with another 25 µL of TEDS, and the supernatant was combined with the eluate from the previous step and diluted with 0.4 mL of DIL. mix. Next, M-280 Streptavidin Dynabeads (prewashed twice with 1 mL of WASH buffer and resuspended in 50 µL of DIL. mix) were added and the mixture was rotated for 30 min at room temperature. The beads were washed three times with 1 mL of WASH buffer, resuspended in 100 µL of TEN buffer , and transferred to a new tube. The beads immobilized on the magnetic stand were rinsed with an additional 1 mL of TEN buffer. On-bead protein digestion followed exactly as described above in the nTAP-MS section.2O at a constant pressure of 80 MPa and separated by a linear gradient of 2%–6% buffer B in buffer A for 3 min, 6%–22% for 40 min, 22%–28% for 9 min, 28%–36% for 8 min, and 36%–80% for 1 min, and 80% buffer B in buffer A for 14 min  on an EASY-Spray column ES801  mounted on a DPV ion source (New Objective) connected to an Orbitrap Fusion (Thermo Scientific) at 150 nL/min flow rate. The xTAP-MS peptides were applied on a µPAC trapping column (PharmaFluidics) in 0.1% formic acid and 2% acetonitrile in H2O at a constant flow rate of 5 µL/min and separated by a linear gradient of 3%–6% buffer B in buffer A for 4 min, 6%–22% for 55 min, 22%–40% for 4 min, and 40%–80% for 1 min, and 80% buffer B in buffer A for 10 min on a 50-cm µPAC column (PharmaFluidics), mounted on an EASY-Spray source (Thermo Scientific) connected to an Orbitrap Fusion Lumos (Thermo Scientific) at 500 nL/min flow rate. Data were acquired using 120,000 resolution for the peptide measurements in the Orbitrap and a top T (3-sec) method with HCD fragmentation for each precursor and fragment measurement in the ion trap following the manufacturer guidelines (Thermo Scientific).Digested peptides were acidified with 0.8% TFA  and analyzed by LC–MS/MS on an EASY-nLC 1000 (Thermo Scientific) with a two-column setup. The nTAP-MS peptides were applied on an Acclaim PepMap 100 C18 trap column  in 0.1% formic acid and 2% acetonitrile in Ht-test.Peptides were identified with MaxQuant version 1.5.3.8 using the search engine Andromeda . The mou5 in 2 mL of S-L-2i medium per well of a six-well plate and transfected with a mixture of 200 ng of plasmid DNA, 0.6 µL of Lipofectamine 3000, and 0.4 µL of P3000 reagent in 100 µL of Opti-MEM medium. After 24 h, cells were rinsed twice with ice-cold PBS and lysed directly in the well with 900 µL of co-IP buffer  supplemented with 1× HALT and, where indicated, also with 1 µL of 10 mg/mL RNase A (Roche). Following 1-h agitation at 4°C, the lysates were collected and centrifuged at 16,000g for 5 min at 4°C. An amount of 2 µL of the cleared supernatants was transferred to a separate tube as the input sample and the rest was mixed with 100 µL of co-IP buffer containing M-280 Streptavidin Dynabeads. Prior to use, the beads  were preblocked for 1 h at room temperature in PBS containing 0.5% cold water fish gelatin (Sigma) and 0.02% Tween-20, and washed twice with co-IP buffer. Following 1-h incubation at 4°C, the beads were separated from the lysate and washed four times with 1 mL of co-IP buffer before resuspending in 15 µL of 1× loading buffer (4× NuPAGE LDS sample buffer and 10× NuPAGE sample reducing agent [Thermo Fisher Scientific] diluted to 1× with H2O). An amount of 13 µL of 1× loading buffer was also added to the input samples. All samples were heated for 3 min at 95°C and subjected to western blotting as described in the Western blotting section below.Cells were seeded 3 × 10g for 3 min. Cell pellets were resuspended in 1 mL of co-IP buffer supplemented with 1× HALT and incubated for 10 min on ice. The lysates were centrifuged at 16,000g for 5 min at 4°C. An amount of 10 µL of the supernatant was transferred to a separate tube as the input sample and stored at 4°C for the duration of the IP. The remaining supernatant was split in two halves, supplemented with 2 µL of anti-2,2,7-trimethylguanosine (anti-m3G) antibody (Sigma) and 1 µL of either 40 U/µL RNasin RNase inhibitor (Promega) or 10 mg/mL RNase A (Roche), and incubated overnight at 4°C with mixing. The next day, 50 µL of Protein G Dynabeads  resuspended in co-IP buffer with 1× HALT was added and incubated for 4 h at 4°C with mixing. Beads were then washed three times with 1 mL of cold co-IP buffer before resuspending in 15 µL of 1× loading buffer. The inputs were mixed with 4 µL of 4× NuPAGE LDS sample buffer and 1.6 µL of 10× NuPAGE sample reducing agent. All samples were heated for 3 min at 95°C and subjected to western blotting as described in the Western blotting section below.Cells were harvested by trypsinization and counted. For each sample, one million cells were rinsed once with PBS and collected by centrifugation at 200g for 3 min. Cell pellets were resuspended in 100 µL of TMNS buffer  containing 25 U of Benzonase and incubated for 5 min at room temperature. An amount of 10 µL of the resulting lysate was mixed with 4 µL of 4× NuPAGE LDS sample buffer and 1.6 µL of 10× NuPAGE sample reducing agent, incubated for 3 min at 95°C and loaded on NuPAGE 4%–12% Bis-Tris Mini Protein Gel (Thermo Fisher Scientific). Proteins were separated by electrophoresis in NuPAGE MOPS SDS running buffer and transferred in Bjerrum and Schafer-Nielsen transfer buffer  onto Immobilon-P PVDF membrane (Millipore), using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) at a constant 15 V for 15 min, followed by a constant 25 V for 25 min. We note that for full-length NRDE2, using the rapid Trans-Blot Turbo Transfer System (Bio-Rad) resulted in suboptimal transfer that cannot be explained by the protein size. For visualization of biotinylated proteins, the membrane was blocked for 15 min at room temperature in TBST containing 1% BSA before adding HRP-Streptavidin (Sigma) to 1:20,000 dilution and incubating for a further 30 min. The membrane was washed three times (5 min each) with TBST, rinsed with PBS, and the proteins were visualized with Immobilon Western Chemiluminescent HRP substrate  using Amersham Imager 600 . For antibody-based western blotting, the membrane was blocked for 10 min at room temperature in TBST containing 1% nonfat dry milk (TBST-NFDM) and then incubated with primary antibody diluted in TBST-NFDM with 0.05% NaN3 overnight at 4°C. The membrane was washed three times (5 min each) with TBST and incubated for 1 h at room temperature with HRP-conjugated secondary antibody diluted 1:10,000 in TBST-NFDM. After that, the membrane was washed, and the signal was developed as described above. When reprobing the same membrane with a different antibody, the membrane was stripped twice for 5 min in 25 mM glycine (pH 2.0), with the first incubation supplemented with 1% SDS, before rinsing thoroughly with water and repeating the blocking and antibody incubations as described above.Cells were harvested by trypsinization and counted. One million cells were rinsed once with PBS and collected by centrifugation at 200Nrde2 (encoding amino acids 2–1172) was probed against Mouse Embryonic Stem Cell_RP1 cDNA library with a total of 58.2 million interactions tested. Gene Ontology terms of 87 high-confidence interactors (PBS score A–D) were analyzed using the clusterProfiler R package  cameras and CFI Plan Apochromat Lambda 100×/1.45 oil immersion objective (Nikon). Fluorescence was excited with 488-nm iBeam Smart (Toptica) and 561-nm Cobolt Jive (Cobolt) lasers, and images (pixel size 0.13 µm) were acquired using VisiView software (Visitron Systems GmbH) with the following settings: 100% laser intensity, 500-msec exposure time, EMCCD GAIN 100 for all mNeonGreen-tagged proteins and 25% laser intensity, 200-msec exposure time, EMCCD GAIN 100 for mCherry-U2AF2. The cells were kept at 37°C and 5% CO2 during all treatments and imaging.Ibidi µ-Slide 8-well chambers were coated with 10 µg/mL Biolaminin LN-511 (BioLamina) diluted in PBS with 1 mM CaCl7 cells were seeded on a 10-cm dish 24 h before cross-linking. For low-abundant proteins, two or four dishes were seeded per sample and processed separately until pooling two at a time during the last FLAG beads wash and, when starting with four dishes, during the last Streptavidin beads wash as described below. Cells were rinsed once on ice and then covered with 5 mL of cold PBS. Open dishes without a lid were placed on an ice-cold aluminum plate in Stratalinker 2400 (Stratagene), and 254 nm UV light was applied for 30 sec to cross-link the protein–RNA interactions. After cross-linking, PBS was removed, cells were scraped in 2 × 0.9 mL of 0.2% BSA in cold PBS and pelleted by spinning at 3000g for 2 min at 4°C. Pellets were resuspended by vortexing in 100 µL of TMS buffer (as in xTAP-MS) prechilled at 12°C and supplemented with 100 U of Benzonase. Following 30-min incubation at 12°C, the lysates were diluted with 0.85 mL of DIL. mix (as in xTAP-MS) and incubated for 5 min on ice. Any remaining insoluble material was removed by spinning at 16,000g for 5 min at 4°C.For each BCLIP-seq sample, 1.5 × 102, 0.02% Tween-20) and transferred in 0.1 mL of 1× PAP to a 0.2-mL PCR tube.The cleared lysates were mixed with anti-FLAG antibody-coupled Protein G Dynabeads resuspended in 50 µL of DIL. mix (as in xTAP-MS) and incubated for 2 h rotating at 4°C, followed by washes, 3xFLAG peptide elution, and incubation with M-280 Streptavidin Dynabeads exactly as described above for xTAP-MS. The Streptavidin beads were washed three times with 1 mL of WASH buffer, once with 0.2 mL of 1× PAP buffer  polymerase (NEB), and incubating for 10 min at 37°C with interval mixing (2000 RPM for 15 sec every 3 min). The reaction was stopped by adding 180 µL of TES buffer  and the beads were washed two more times with 0.2 mL of TES before resuspending in 20 µL of TET buffer  with 0.5 µL of 20 mg/mL Proteinase K (Roche). After 30 min of protein digestion at 50°C with interval mixing, Proteinase K was inactivated for 3 min at 85°C. The beads were separated on a magnet, and the supernatant was transferred to a new tube and mixed with 20 µL of homemade SPRI beads  and 40 µL of isopropanol. RNA was precipitated rotating for 10 min at room temperature, beads were separated and rinsed on a magnet twice with 0.2 mL of 80% ethanol and dried for 1 min, and finally RNA was eluted in 15.25 µL of 0.02% Tween-20.The purified cross-linked RNA fragments were polyadenylated by resuspending the beads in 20 µL of 1× PAP buffer containing 0.1 mM ATP and 2.5 U 2, 20 mM TCEP), 0.5 µL of RNase Block (Agilent), and 0.25 µL of SuperScript II (Thermo Fisher Scientific) and incubating for 15 min at 42°C, after which 1 µL of 40 mM MnCl2 and 1µL of 20 µM TSO were added, and the 42°C incubation continued for another 30 min followed by a 15-min heat inactivation at 70°C. The reaction was slowly cooled down to room temperature with a Δ −1°C/sec gradient, and 0.5 µL each of 5 U/µL RNase H (NEB), 1000 U/µL RNase T1 (Thermo Fisher Scientific), and 20 U/µL Exonuclease I (NEB) were added to degrade the excess TSO and RT primer for 20 min at 37°C, followed by a 15-min heat inactivation at 80°C. The resulting cDNA was purified by isopropanol precipitation on SPRI beads as described above and eluted in 17.2 µL of 0.02% Tween-20.The purified RNA was mixed with 1 µL of 0.2 µM RA3dT18V primer and 1 µL of 10 mM dNTPs , denatured for 3 min at 65°C, and quickly cooled on ice. Reverse transcription was started by mixing with a master mix consisting of 2 µL of 10× RT buffer .To amplify the cDNA library, the eluates from the previous step were transferred to a 0.2-mL low-profile PCR tube (Bio-Rad) and mixed with a master mix consisting of 20 µL of 2× NEBNext Ultra II Q5 Master Mix (NEB), 1 µL of 40 µM P5* primer, 1 µL of 40 µM PE* primer, and 0.8 µL of 100 µM EvaGreen Fluorescent DNA Stain (Jena Bioscience). The reactions were amplified in a CFX96 Real-Time PCR instrument (Bio-Rad) with an initial denaturation for 45 sec at 98°C, followed by cycles of 10 sec at 98°C and 50 sec at 65°C, plate read, and10 sec at 65°C. The amplification was monitored in real time, and the reactions were removed after three cycles of exponential fluorescence signal increase . The amplified library was purified by mixing with 32 µL of homemade SPRI beads and incubating for 5 min at room temperature. The beads were then separated and rinsed on a magnet twice with 0.2 mL of 80% ethanol and dried for 1 min, and DNA was eluted in 20 µL of HSupplemental Table S3.All BCLIP-seq related oligonucleotides were ordered as standard desalted RNA/DNA oligos or ultramers from IDT and their sequences are listed in Total RNA was isolated from near-confluent 6-cm dishes using the Absolutely RNA Miniprep kit (Agilent), including an on-column DNase treatment step, according to the manufacturer's instructions. Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat kit (Illumina), including a ribosomal RNA depletion step, and sequenced on the Illumina HiSeq2500 platform (50-nt single-end reads).Ct method and normalized to Actb expression. All qPCR primer sequences are listed in Supplemental Table S4.Total RNA was isolated as described in the RNA-seq section and 500 ng was reverse-transcribed using the PrimeScript RT Master Mix (Takara). A volume of cDNA corresponding to 5 ng of the input RNA was subjected to quantitative PCR (qPCR) in a 10-µL reaction using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.4 µM each forward and reverse primer. The qPCR was performed in a CFX96 Real-Time PCR instrument (Bio-Rad) with an initial denaturation for 30 sec at 95°C, followed by 40 cycles of 5 sec at 95°C,10 sec at 60°C, and plate read. The relative RNA levels were determined using the ΔΔFor the splicing analysis of endogenous transcripts, cells were grown to near confluency on 6-cm dishes and total RNA was extracted using the RNAzol RT reagent (Sigma) following the manufacturer's protocol. An amount of 1 µg of the RNA was reverse-transcribed in a 10-µL reaction using SuperScript IV (Thermo Fisher Scientific) and random primers (Agilent), and a volume of cDNA corresponding to 5 ng of the input RNA was subjected to 30 cycles of PCR with the 2× NEBNext Ultra II Q5 Master Mix (NEB).5 wild-type cells or 4 × 105Nrde2-KO cells (2 d of 4OHT treatment) were seeded on a six-well plate, transfected with 500 ng of the corresponding reporter plasmid DNA using Lipofectamine 3000, and grown for 48 h. Cells were harvested in 0.5 mL of RNAzol RT reagent supplemented with 1 µL of Polyacryl Carrier (MRC), and total RNA was extracted following the manufacturer's protocol. An amount of 500 ng of the RNA was treated with ezDNase (Thermo Fisher Scientific) and reverse transcribed using SuperScript IV and random primers. A volume of cDNA corresponding to 2.5 ng of the input RNA was subjected to 35 cycles of PCR with the 2× NEBNext Ultra II Q5 Master Mix (NEB).To determine the splicing pattern of the luciferase reporter transcripts, 2 × 10Supplemental Table S4.The PCR reactions were purified using homemade SPRI beads (as described in the BCLIP-seq section), resolved on a 1% agarose gel containing Midori Green Advance stain (Nippon Genetics) and visualized using the FastGene FAS-DIGI PRO gel imaging system (Nippon Genetics). The sequences of primers used for splicing pattern analysis are listed in 6 cells (3 d of 4OHT treatment for Nrde2-KO cells) were seeded on a 6-cm dish and grown overnight. Cells were rinsed and scraped in 2 × 0.5 mL of cold PBS on ice and pelleted by spinning at 3300g for 2 min at 4°C. The pellet was resuspended by pipetting in 0.2 mL of Ribo-seq buffer  and incubated for 5 min on ice, followed by spinning at 7600g for 5 min at 4°C. An amount of 100 µL of the supernatant was transferred to a new tube, mixed with 100 µL of H2O and 0.4 µL of 250 U/µL Benzonase, and incubated for 30 min at 37°C with constant shaking. The nuclease digestion was stopped by adding 0.5 mL of RNAzol RT reagent with 1 µL of Polyacryl Carrier, and RNA fragments were purified following the manufacturer's total RNA isolation protocol. The precipitated RNA was resuspended in 5 µL of H2O, mixed with 5 µL of 2× Novex TBE-Urea sample buffer (Thermo Fisher Scientific) and denatured for 3 min at 70°C. RNA was resolved on a Novex 15% TBE-Urea gel (Thermo Fisher Scientific) in 1× TBE buffer for 75 min at 180 V side by side with 20 ng of a 35-nt-long single-stranded DNA oligo 5′-ACCACTCGAGTCAAAACAGAGATGTGTCGAAGATG-3′ (IDT). The gel was stained in 20 mL of 1× TBE buffer containing 2 µL of SYBR Gold stain (Thermo Fisher Scientific) for 5 min at room temperature, and a band migrating just above the 35-nt oligo was cut from the Ribo-seq sample lane.For each Ribo-seq sample, 2 × 10g for 5 min at room temperature. RNA was precipitated by adding 1 µL of glycogen (Roche) and 1 mL of ethanol to the flow-through and incubating for 1 h at −20°C. RNA was pelleted by spinning at 16,000g for 15 min at 4°C, and the pellet was washed twice with 0.5 mL of cold 80% ethanol, dried for 5 min at room temperature, and finally resuspended in 10 µL of C1 buffer .The gel piece was submerged in 100 µL of 400 mM NaOAc (pH 5.2) and frozen at −80°C. After thawing the gel-containing tube for 5 min at 95°C, the gel piece was crushed with a plastic pestle, 300 µL of 400 mM NaOAc (pH 5.2) was added, and RNA was extracted from the gel by three cycles of 5-min incubation at 95°C and 20-min shaking at 22°C. The mixture was cleared by spinning through a Corning Costar Spin-X filter  at 20,000Before the next step, custom Ribo-beads were prepared as follows. Streptavidin MyOne C1 Dynabeads (Thermo Fisher Scientific) equivalent to 20 µL of the original slurry were washed twice with 0.2 mL of C1 buffer and resuspended in 20 µL of C1 buffer. Two rRNA depletion oligos were ordered from IDT as 3′-biotinylated standard desalted custom DNA oligos CCGTACGCCACATTTCCCACGCCGCGACGCGC/3BioTEG/ and CAAGACGAACGGCTCTCCGCACCGGACCCCGGTCCC/3BioTEG/, resuspended, and mixed to 50 µM each. Before the first use, the mixture was denatured for 1 min at 95°C and quickly cooled on ice. An amount of 2 µL of the rRNA depletion oligo mix was added to the 20 µL of C1 Dynabeads and incubated for 15 min at room temperature with occasional mixing. The beads were washed twice with 0.2 mL of C1 buffer and resuspended in 20 µL of C1 buffer.The purified RNA fragments were mixed with 10 µL of the Ribo-beads and incubated for 3 min at 70°C, followed by 10 min at 50°C. Beads were separated on a magnet, and the supernatant was mixed with the remaining 10 µL of the Ribo-beads. After another round of 3 min at 70°C and 10 min at 50°C, the supernatant was mixed with 30 µL of homemade SPRI beads (as in BCLIP-seq) and 50 µL of isopropanol, and the RNA was precipitated rotating for 10 min at room temperature. The beads were separated, rinsed on a magnet twice with 0.2 mL of 80% ethanol, and dried for 1 min, and finally RNA was eluted in 13.5 µL of 0.02% Tween-20.E. coli Poly(A) polymerase (NEB) and incubated for 10 min at 37°C with interval mixing (2000 RPM for 15 sec every 3 min). The reaction was stopped by the addition of 20 µL of homemade SPRI beads and 40 µL of isopropanol. Subsequent polyadenylated RNA purification, reverse transcription with template switching, cDNA library amplification, and sequencing on Illumina HiSeq2500 were performed exactly as described in the BCLIP-seq section.The purified rRNA-depleted RNA fragments were polyadenylated by adding 4 µL of 5× PAP buffer (as in BCLIP-seq), 2 µL of 1 mM ATP, and 0.5 µL of 5 U/µL g for 2 min. The pellets were washed once and resuspended in cold PBS. The mNeonGreen fluorescence of the endogenously expressed 2C reporter was measured on a BD LSR II flow cytometer (BD Biosciences) using a 488 nm excitation laser and a FITC filter. The acquired data were analyzed using the FlowJo software.Cells from a near-confluent six-well plate were harvested by trypsinization and pelleted by spinning at 300Emg1 and Phb2 genes, into the pBluescript II KS-plasmid (Agilent Technologies). For in-frame fusion with Cdk2 and Tti1 fragments, a P2A self-cleaving peptide sequence was included to separate the Renilla luciferase. Site-directed mutagenesis was performed by PCR with NEBNext High-Fidelity 2× PCR master mix (NEB) using overlapping primers with 15-nt overhangs carrying the desired mutation (IDT).The backbone control luciferase reporter plasmid was created by placing the Renilla and Firefly luciferase coding sequences with SV40 and BGH polyA sites, respectively, under the control of a minimal endogenous bidirectional promoter driving the expression of mouse 5 to 6 × 105 per well of a 96-well plate and transfected with 10 ng of the corresponding reporter plasmid supplemented with 90 ng of pBluescript II KS, using Lipofectamine 3000. Nrde2-KO cells were treated with 4OHT for 2 d prior to transfection. Where appropriate, the cells were treated with the dTAG-13 compound 24 h post-transfection. After 48 h post-transfection, cells were washed with PBS and lysed in 25 µL of Passive Lysis Buffer (Promega) for 20 min at room temperature. An amount of 10 µL of the lysate was used to measure luciferase activity by sequential mixing with 10 µL of each substrate from the Dual-Luciferase Reporter Assay System (Promega). Luminescence was measured on a Mithras LB 940 microplate reader (Berthold Technologies). All Renilla luminescence values were first normalized to Firefly and the normalized activity was then calculated relative to the empty control reporter in wild-type cells.Cells were seeded 2 × 107 cells were collected by centrifugation at 300g for 2 min and rinsed once with 1 mL of PBS. Cell pellets were resuspended in 1 mL of cold co-IP buffer  supplemented with 1× HALT in a 15-mL Bioruptor tube containing one scoop of sonication beads (Diagenode) and sonicated in Bioruptor Pico (Diagenode) in five cycles of 30 sec ON/30 sec OFF. Lysates were cleared by centrifugation at 16,000g for 5 min at 4°C, and 1 µL of the supernatant was taken to a separate tube as 0.1% input. The remaining supernatant was mixed with anti-FLAG antibody-coupled Protein G Dynabeads . After 2 h rotating at 4°C, the beads were washed three times with 1 mL of cold co-IP buffer, and FLAG-tagged proteins were eluted by two consecutive 15-min incubations with shaking (500 RPM) at room temperature—first in 100 µL of co-IP buffer with 1× HALT containing 0.25 mg/mL 3xFLAG peptide, and second in 25 µL of co-IP buffer with 1× HALT containing 1 mg/mL 3xFLAG peptide. The beads were rinsed with another 325 µL of co-IP buffer, and the supernatant was combined with the eluates from the previous step. M-280 Streptavidin dynabeads (prewashed twice with 1 mL of co-IP buffer) resuspended in 50 µL of co-IP buffer with 1× HALT were then added, and the samples were incubated for 30 min at room temperature with mixing. Beads were washed on ice three times with 1 mL of cold co-IP buffer and resuspended in 0.5 mL of RNA extraction buffer , which was also added to the inputs. RNA was extracted by adding 0.5 mL of buffered phenol:chloroform:IAA  and shaken for 1 min, followed by spinning at 20,000g for 5 min at room temperature. The upper phase was transferred to a new tube, and RNA was precipitated by mixing with an equal volume of isopropanol in the presence of 1 µL of Polyacryl Carrier (MRC). After 10-min incubation at room temperature, RNA was pelleted by centrifugation at 16,000g for 30 min at 4°C, washed once with 0.5 mL of 75% ethanol, air-dried for 5 min, and resuspended in 5 µL of water. Next, 5 µL of 2× Novex TBE-Urea Sample Buffer (Thermo Fisher Scientific) was added, and samples were denatured for 3 min at 95°C and immediately cooled on ice. RNA was separated on a 6% Novex TBE-Urea gel (Thermo Fisher Scientific) for 70 min at 90 V in 1× TBE buffer preheated to 50°C. The gel was briefly rinsed with 1× TBE, and RNA was transferred to positively charged Nylon membrane (Roche) using Fastblot (Biometra) for 30 min at a constant current of 200 mA. After the transfer, RNA was cross-linked to the wet membrane for 90 sec using Stratalinker 2400. The membrane was air-dried, washed in 2× SSC buffer containing 0.1% SDS, and prehybridized in 10 mL of ULTRAhyb-Oligo buffer (Thermo Fisher Scientific) for 90 min at 37°C. Twenty picomoles of a 3′-end biotinylated ssDNA oligo (GTATCTCCCCTGCCAGGTAAGTAT/Bio/ for U1 snRNA and TACTGCAATACCAGGTCGATGCGT/Bio/ for U2 snRNA)   was thenSupplemental Figure S3A,C  to the mouse genome (mm10), including the transcriptome annotation of GENCODE release M23 using STAR version 2.7.0a , with thFor BCLIP-seq read categorization B, noncolTo analyze snRNA-mapping reads, noncollapsed reads were aligned to snRNA sequences using Bowtie2 version 2.3.5.1 with –sensitive parameters. Reads that were at least 20 bp long and mapped with less than 1 edit distance (NM:i:0 or NM:i:1) were used to calculate coverage across snRNAs.RNA-seq reads were aligned (splicing-aware) to the mouse genome (mm10), including the transcriptome annotation of GENCODE release M23 using STAR version 2.7.3a with the parameters –outFilterMultimapNmax 100, –outFilterMismatchNoverLmax 0.05, and –outSAMmultNmax 1.GENCODE release M23 annotation was used for all analysis unless otherwise stated. The number of reads in all genes for all WT samples were counted using featureCounts from the Rsubread package  with theAll genes with FPKM > 0 were used as expressed genes. In addition, transcript abundance was quantified using Salmon version 1.2.0  with thehttps://github.com/fmi-basel/gbuehler-MiniChip): Coverage was calculated in regions ±200 bp around all 5′SSs, using only uniquely mapping reads to the forward strand, and displayed as the average cpm across two replicates.Heat maps were generated using the MiniChip R package  were removed. Each BCLIP-seq .bam file was loaded using RSamtools R package, and the reads that overlap 5′SSs were selected. Spliced or unspliced reads were counted separately for each junction and each sample .bam file. Then the 5′SSs with spliced and unspliced read counts above 10 were selected, and the fold change between spliced and unspliced read counts was calculated.P-value <0.01 and log2FC >1/−1 were counted as up-/down-regulated.The number of correctly stranded uniquely mapping RNA-seq reads was quantified in genes using featureCounts (Rsubread package). CHX-treated and untreated samples were analyzed separately using DEseq2 , first flimma R package  was then calculated.Repeat expression was quantified with featureCounts using RepeatMasker tracks for LTR, LINE, SINE, and DNA elements, removing repeats that overlap genes, using only uniquely mapping reads. The counts were normalized using the TMMwsl method from the edgeR package , taking  package  was usedThe number of correctly stranded uniquely mapping RNA-seq reads was quantified in expressed introns using featureCounts with the parameters useMetaFeatures = FALSE, allowMultiOverlap = TRUE, minOverlap = 10, countMultiMappingReads = FALSE, fraction = FALSE, minMQS = 255, and strandSpecific = 2.P-value <0.05 and log2FC >0.5/−0.5 were counted as up/down-regulated. Finally, up-regulated introns within genes that are not up-regulated (log2FC < 0) were selected for further analysis.CHX-treated and untreated samples were analyzed separately using DEseq2, first filtering out introns that have less than 110 counts across all samples or 40 counts across all CHX samples. Introns with an adjusted All intron features  were calculated using Matt . Fastq fCLIPper tool  was usedRibo-seq reads were preprocessed as described for BCLIP-seq. Read mapping and differential gene expression analysis for Ribo-seq data were performed as for RNA-seq data. Genes with less than 10 reads across all samples were excluded from differential expression analysishttps://data.mendeley.com/datasets/v24ytccs4h/2. Custom scripts and tools for data analysis are available on GitHub (https://github.com/xxxmichixxx/Nrde2Project) or are indicated in the respective Materials and Methods sections.All reagents and cell lines generated in this study are available from the corresponding author, Marc Bühler (marc.buehler@fmi.ch), upon a Materials Transfer Agreement. All sequencing data generated in this study have been deposited at NCBI GEO and are available under accession number GSE179744. Mass spectrometry data have been deposited at ProteomeXchange via the PRIDE database under accession number PXD029392. Original western and northern blot images have been deposited at Mendeley Data and can be accessed at Supplemental material is available for this article.The Friedrich Miescher Institute for Biomedical Research (FMI) receives significant financial contributions from the Novartis Research Foundation. Published research reagents from the FMI are shared with the academic community under a Material Transfer Agreement (MTA) having terms and conditions corresponding to those of the UBMTA .


We then provide the structural basis of this function with a high-resolution cryo-EM structure of nsP1 in complex with the unconventional cap-1 substrate RNA m7GpppAmU. We show that the 5′ppRNA species generated by decapping can trigger retinoic acid–inducible gene I–mediated interferon response. We further demonstrate that the decapping activity is conserved among the alphaviral nsP1s. To our knowledge, this is a new mechanism through which alphaviruses activate the antiviral immune response. This decapping activity could promote cellular mRNA degradation and facilitate viral gene expression, which is functionally analogous to the cap-snatching mechanism by influenza virus.Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) contains both the N7-guanine methyltransferase and guanylyltransferase activities and catalyzes the 5′ end cap formation of viral RNAs. To further understand its catalytic activity and role in virus–host interaction, we demonstrate that purified recombinant CHIKV nsP1 can reverse the guanylyl transfer reaction and remove the m Cryoby GTase .In eukaryotes, the decapping process is the reverse of the capping reaction as part of the mRNA degradation pathway to regulate mRNA gene expression where the 5′ cap is removed where thereby allowing targeting by exoribonuclease in the 5′-3′ pathway. Uncapped RNAs such as viral transcripts are targets for degradation by host enzymes and may trigger antiviral innate immune responses. The host antiviral response is activated through the expression of interferon-stimulated genes (ISGs), which target viral RNAs through sensing by cytoplasmic pattern recognition receptors such as retinoic acid–inducible gene I (RIG-I) and MDA5. They detect dsRNA viral replicative intermediates, molecular features associated with viral genomes, or the absence of features associated with host mRNAs, resulting in degradation and repression of viral RNA expression. As such, viruses have several strategies to acquire the 5′ cap structure . They caSaccharomyces cerevisiae. In this scenario, decapping can function to overcome the disadvantages of uncapped viral transcripts as the L-A capsid Gag protein can form a covalent bond between m7Gp and H154, resulting in the removal of mRNA cap from cellular mRNA. This results in increased viral mRNA expression through the generation of capless cellular mRNAs, which act as decoys for the cellular mRNA decay system  signaling through the generation of 5′ diphosphate RNA by-products as a result of CHIKV nsP1–mediated decapping.Here, we demonstrate the decapping activity of CHIKV nsP1, through A). First, CHIKV nsP1 was able to snatch the m7Gp moiety from several biological and chemical sources, such as total RNA and mRNA extracted from Expi293 cells, m7GpppA, and capped RNA m7GpppAU-10. The formation of m7Gp-nsP1 covalent intermediate was detected by immunoblotting of m7Gp moiety on nsP1 protein (B). Next, we show that the m7Gp-nsP1 covalent intermediate could successfully transfer m7G to a suitable RNA substrate (5′ pppAU-10 RNA oligo) and result in capped RNA product, despite varying efficiency (C above). Cap-0 donors such as m7GpppAU-10, m7GpppAU-10 Alexa 647 , as well as short-capped RNAs m7GpppAmU and m7GpppAmG  could promote the capping of 5′ pppAU-10 RNA oligo. Using 150 ng of total RNA and mRNA each did not  result in detectable capping, likely due to the heterogeneity of RNA length, low concentration or both. We further quantified the capping reactions by analyzing the band intensity, which is proportional to the amount of capped RNAs product formed . Previous work demonstrated sequence preference for the capping  is preferred over m7GpppAmG . There is more capped RNA product formation when m7GpppAmU is the donor  . As such, the m7GpppAm (Cap-1) structure can serve as an effective m7Gp donor as there is no clash for a methylated 2′ hydroxy group (2′-O-me) on the A1 ribose sugar of m7GpppAU-10. This suggests that nsP1 can cleave off the m7Gp from both Cap-0 and Cap-1 RNA, resulting in the formation of the Cap-0 RNA product. This highlights the functional versatility of nsP1 as it can potentially extract the m7Gp from a variety of capped RNAs.To examine if the RNA 5′end capping reaction may be reversible, we measured CHIKV nsP1 capping/decapping activity A. First, protein B. Next, ficiency C above.  capping  which maA). We expressed CHIKV nsP1 WT and the catalytically inactive mutant H37A (nsP1-mut) in A549 Dual and A549-Dual KO-RIG-I cells (B). The IRF3 pathway is significantly more stimulated when nsP1 wt is expressed in A549 Dual than nsP1-mut and the vector control  as well as comparing IRF3 activation in A549 Dual and KO-RIG-I when nsP1 is expressed. These results indicate that active nsP1 indeed activates type I IFN immune response. In contrast, when expressed in the KO-RIG-I cells, no significant changes to the luciferase activities could be detected. This suggests that the IFN response is mediated through the RIG-I signaling pathway.We hypothesized that the decapping activity of nsP1 could occur within host cells given there are plenty of host mRNAs. The consequence is the production of 5′ diphosphorylated RNAs, which may in turn stimulate RIG-I–mediated IFN signaling. We then tested this hypothesis by expressing CHIKV nsP1 in the reporter A549 Dual and a RIG-I KO A549-Dual KO-RIG-I cell lines. They allow monitoring of the interferon regulatory factor 3 (IRF3) pathway through the activity of ISG54 promoter-driven Lucia luciferase A. We exp-I cells B. The IRt test p-value <0.0189 VEEV 95% CI −480.2 to −57.37; Unpaired t test p-value <0.0076 SINV 95% CI -816.2 to −173.0). In addition, the IRF3 pathway is significantly more stimulated when wt nsP1 from both VEEV and SINV  was expressed in A549 Dual compared to RIG-I KO cell line A549-Dual KO-RIG-I cells (C). Together, this suggests the presence of decapping enzymatic activity in other alphavirus nsP1s can potentially activate IFN and it is mediated by RIG-I. Interestingly, we also observed some IFN activity in the nsP1 mutants, suggesting there is also activation independent of catalytic activity.Alphaviral nsP1s are highly conserved , 6, 12 s-I cells C. Togetht test p-value <0.05; total RNA (AP) 95% CI 213.5–8176; total RNA 95% CI 1047–6813) (A). The 3p10LA9 was used as a positive control as it specifically activates RIG-I  A. The 3pes RIG-I .Figure 3t test p-value <0.01; 95% CI –1600 to −544.4) (B) Similar to previous experiments, the IRF3 pathway is significantly more stimulated when nsP1 is expressed in A549 Dual than nsP1-mut . In addition, there was IRF3 pathway activation when in A549 Dual tet-induced expression of CHIKV nsP1-mut cells were subsequently transfected with a plasmid containing overexpression of wt CHIKV nsP1 compared to the KO-RIG-I cells and a control vector  (C). Together, we observe that there is both catalytic-dependent and catalytic-independent stimulation of the IFN activation and response, where the former is likely a result in 5′ppRNA production, which subsequently activates RIG-I–mediated innate immune response in the host  with the induction of tetracycline. The IRF3 pathway is significantly more stimulated after tet-induced CHIKV nsP1 expression in A549 Dual compared to A549-Dual KO-RIG-I (unpaired  −544.4) B Similar −12828) C. Togeththe host .7GpppAmU at a resolution of 2.4 Å by cryo-EM . The guanine base and ribose ring of the m7G0 cap are located at the same position as that of the previously reported nsP1-m7GTP structure . The guanine base, bound to the pocket formed by residues D152, Y154, Y248, and E250, forms stacking interactions with the aromatic side chain of Y154 and Y248 and is further stabled by a hydrogen bond (H-bond) with E250. The ribose ring makes distant van der Waals interaction with V243 and forms an H-bond with the side chains of D152 and Y285. Further, the triphosphates of m7G0 cap form H-bonds with the positively charged side chains of arginine residues, R41 and R70 . The α- and β-phosphates interact with a magnesium ion, while the γ-phosphate indirectly interacts with a magnesium ion in a water-mediated manner. The m7G0 cap was captured in a preguanylyl-transferring state, explaining the importance of the magnesium coordination to its α- and β-phosphates at the GTase active site.To reveal the decapping mechanism of nsP1, we determined the capping/decapping inactive mutation H37A nsP1-mut in complex with SAH and mRNA analog, Cap-1 RNA m cryo-EM . The oveD 1.22 Å ). All tw and SAH A. The guPDB 7FGG ) . The A1 base is stabilized by the T33-P34-N35 loop and V279 of the neighboring nsP1. The A1 base also forms H-bond with the backbone of V279 (neighboring nsP1-mut and water-mediated H-bonds with N35 and D36). The 2′-O-me group of A1 ribose forms van der Waals interaction with T33. The U2 base is sandwiched between the A1 base and the side chain of R289 from the neighboring nsP1(H37A). Another residue E54 from the neighboring nsP1-mut forms polar interaction with it. The phosphate between A1 and U2 forms an H-bond with the side chain of K99. Compared to our reported structure of nsP1-mut in complex with Cap-0 RNA m7GpppAU-10 . This conformation may be due to the absence of nucleotides after U2, which does not seem to affect the decapping activity.The nucleotides A1 and U2 of Cap-1 RNA, stacked on to each other, are both located in the groove between the neighboring nsP1-mut molecules C. The A1pppAU-10 , the m7Gper ring D. This c7GTP-bound nsP1  structural rearrangement occurs at the triphosphates moiety of m7GpppAmU, where (2) the α- and β-phosphates move closer towards H37 (α-phosphate to H37 distance reduced to ∼3.2 Å) coordinated with magnesium ion and water molecules (B and C). The orientation differences of the capped-RNA substrate binding to the nsP1 pocket could be due to the short capped RNA presented here. which favors the typical base stacking conformation and is stabilized by neighboring nsP1 residues, R289 and E54 (C and D). Overall, nsP1 is able to transfer the m7Gp moiety from capped mRNA to the diphosphorylated 5′ end of viral transcript thus generating an authentic cap structure. These findings are consistent with previous work demonstrating that nsP1 can release significant amounts of intermediates of the reaction and can decap RNA substrates  to modeolecules , B and C and E54 , C and Dbstrates . They albstrates .A)  , 21, 22 A) . L-A virA) , 24. Whiactivity  resultinactivity . This taactivity .Figure 57GpppAmU is preferred over m7GpppAmG (D) unsurprising seeing as capping also demonstrates sequence preference (B), a mRNA decapping enzyme that is a member of the histidine triad (HIT) family of hydrolases which is involved in the removal of cap following mRNA decay . This also has potential implications for alphavirus-based self-amplifying RNAs, which utilize the nsPs as the functional replicase which could restrict self-amplifying RNAs amplification and antigen expression . Subsequently, as the infection progresses resulting in virus-mediated transcription inhibition and host translational shutoff to dampen IFN antiviral response. Finally, the formation of the mature replication complex and synthesis of +ssRNA triggers nsP1 to switch to viral RNA capping before exporting it into the cytosol for the packaging and formation of mature virions.Combining with the recently elucidated CHIKV replication complex gives insight that the viral replication is tightly protected in the spherule, allowing for ssRNA capping to be orchestrated by nsP1 and nsP2 before exporting it into the cytosol , 39 whicFig. 5The main limitation of this method to evaluate RNA decapping activity may not reflect viral infection and replication. It omits the role of the other nsPs and host factors during viral replication after infection and may overemphasize the decapping activity, when decapping might be a rare occurrence in early viral infection, to prevent translational overload, when GTP/SAM is depleted or to maintain the capped/uncapped RNA ratio to regulate the virus replication cycle. Future work may be to systemically dissect nsP1-mediated virus-host integration pathways and their role in viral infection and replication.et al (WT and mutant nsP1 H37A (hereafter referred to as nsP1-mut) were expressed and purified as described in the study by Zhang et al .et al. was used with modifications . The reactions were incubated at 30 °C for 12 h and terminated by adding the stop solution 2× RNA loading dye . The capped RNA products were separated by 20% denaturing RNA 8M urea PAGE gel and visualized using ChemiDoc MP imaging system (Bio-Rad). The bands were quantified with Image Lab  and plotted and analyzed on Prism 9.3.1 .The method previously described by Li ications . CHIKV 5g for 5 min at 4 °C and the supernatant was removed. The pellet was resuspended in PBS and incubated with TRIzol reagent (Thermo Fisher Scientific). Chloroform was added and the mixture was spun at 12,000g for 30 min at 4 °C to separate the aqueous and organic phase. Total RNA was extracted from the aqueous phase after the addition of isopropanol and washing with ethanol. The pellet is spun for an additional 10 min at 7,500g at 4 °C, the ethanol was removed and air-dried. The pellet is resuspended in the desired amount of RNase-free water (Hyclone), and the concentration is determined by spectrophotometry. The RNA sample is denatured at 65 °C with RNA loading dye and run on a 0.6% agarose gel at 100 V for 1 h for visualization. For mRNA extracts, total RNAs are pooled and further purified using the Oligotex mRNA kit (Qiagen) as per the manufacturer’s instructions.Expi293 cells in suspension were grown, pellet at 2002 and 0.5 mM SAM and 1 mM GTP or alternative cap donors such as mRNA or total RNA extracts and incubated for 2 h at 30 °C. It was stopped with the addition of Laemmli sample buffer, and products were loaded onto a 12% SDS-PAGE gel. The 12% SDS-PAGE gel was stained with Coomassie blue or prepared for immunoblotting and transferred onto a polyvinylidene difluoride membrane (Sigma-Aldrich) with 1× Transfer buffer (48 mM Tris 39 mM glycine and 20% methanol) by Trans-Blot Turbo (Bio-Rad) for 20 min at max 2.5 A and 25 V. The membrane was blocked with 5% bovine serum albumin 1× PBS for 30 min at room temperature and subsequently incubated with the primary antibody anti-7-m7G cap  1 μg/ml overnight at 4 °C and washed thrice with washing buffer 1% bovine serum albumin 1× PBS 0.1% Tween-20 for 5 min with agitation. The secondary antibody  is added to the membrane for 1 h at room temperature and washed thrice again with washing buffer before the addition of the chemiluminescence reagent . The extra reagent is removed from the membrane and visualized on the ChemiDoc MP imaging system (Bio-Rad).Reactions were prepared in 20 μl containing 1.5 μM nsP1, 50 mM Tris–HCl pH 7.5, 2 mM DTT, 10 mM KCl, and 2 mM MgCl2 incubated at 30 °C for 2 h. Subsequently, the reactions were then incubated with TRIzol reagent (Thermo Fisher Scientific). Chloroform was added and the mixture was spun at 12,000g for 30 min at 4 °C to separate the aqueous and organic phase. Total RNA was extracted from the aqueous phase after the addition of isopropanol and washing with ethanol. The pellet is spun for an additional 10 min at 7500g at 4 °C, the ethanol was removed and air-dried. The pellet is resuspended in the desired amount of RNase-free water (Hyclone), and the concentration is determined by spectrophotometry. The RNA sample is denatured at 65 °C with RNA loading dye and run on a 0.6% agarose gel at 100 V for 1 h for visualisation.To generate dephosphorylated 5′ RNA, total RNA was treated with alkaline phosphatase (Promega) 100 μl reaction was set up as per manufacturer’s instructions. To generate CHIKV nsP1-decapped RNA, recombinant CHIKV nsP1 treated total RNA, 100 μl reaction was set up with 30 mg total RNA added to 20 μM recombinant CHIKV nsP1 in 50 mM Tris–HCl pH 7.5, 2 mM DTT, 10 mM KCl, and 2 mM MgClin vitro using T7 RNA polymerase as described previously , 1 μm DNA template (IDT), 400 to 600 nm T7 RNA polymerase, 0.2 U·mL−1 thermostable inorganic pyrophosphatase (New England Biolabs) for overnight at 37 °C. Phenol:chloroform:isoamyl alcohol (25: 24:1) (Merck) was used to stop in vitro transcription reactions and extract RNA. The RNA was precipitated overnight at −80 °C by adding three volumes of ethanol 95% in the presence of 0.1% (v/v) of sodium acetate. The RNAs were further isolated by Hi-Trap Q HP column and excised from 20% denaturing urea-PAGE. The quality of expected RNAs was determined again on 20% denaturing urea-PAGE prior to downstream experiments.RNA 3p10LA9 pppGGAUUUCCACCUUCGGGGGAAAUCC were transcribed eviously , 41. The2 incubator. A549 Dual and its KO-RIG-I equivalent cells express secreted Lucia luciferase reporter gene under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements. Both cell lines were grown on 96-well plates and were first transfected with CHIKV nsP1 expression plasmid (pCMV-CHIKV nsP1) or its catalytically inactive mutant H37A (nsP1-mut) by PEI MAX (Polysciences) for transient expression for 24 h. Subsequently, the supernatant was collected, and luciferase activity is measured by QUANTI-Luc reagent (as per manufacturer’s instructions) onto a 96-well plate (Corning) with a microplate reader (Synergy H1 BioTek). The luciferase activity is normalized to cells transfected with a control vector pBad His6 MBP TEV LIC (Addgene #37503) and cells that were not transfected. Each experiment was performed in triplicate and replicated. The results were analyzed with an unpaired t test on Prism 9.3.1 (GraphPad). The experiment was performed similarly with VEEV nsP1 and its inactive H37A (VEEV nsP1-mut) mutant as well as SINV nsP1 and its inactive H39A (SINV nsP1-mut) mutant.A549 Dual and A549 Dual KO-RIG-I cells (InvivoGen) were maintained in Dulbecco’s Modified Eagle Medium (Gibco) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin at 37 °C in a 5% COt test on Prism 9.3.1 (GraphPad).Both cell lines were grown on 96-well plates were first transfected with 100 ng of treated RNA with LyoVec (InvivoGen). After 48 h, the supernatant was collected, and luciferase activity is measured by QUANTI-Luc reagent (as per manufacturer’s instructions) onto a 96-well plate (Corning) with a microplate reader (Synergy H1 BioTek). The controls were cell lines transfected with untreated total RNA and hairpin RNA 3p10LA9. The results were analyzed with an unpaired The CHIKV nsP1 WT and H37A mutant genes were cloned into the vector pInducer20 (Addgene #44012), pInducer CHIKV nsP1, and pInducer CHIKV nsP1-mut, respectively. Lentiviral supernatants were generated by transfection of HEK293T cells according to Mirus Bio's TransIT transfection protocols and pooled 96 h after transfection. Lentiviral titers were measured with Lenti-X GoStix Plus (Takara Bio) and concentrated with Lenti-X concentrator as per the manufacturer’s instructions. The lentivirus was resuspended in PBS and stored at −80 °C until required.D). A549 Dual CHIKV nsP1, A549 Dual KO-RIG-I CHIKV nsP1, A549 Dual CHIKV nsP1-mut, and A549 Dual KO-RIG-I CHIKV nsP1-mut were grown on 24-well plates. Cells were washed with 1× PBS (Gibco), and media was replaced with Dulbecco’s Modified Eagle Medium (Gibco) containing 10% tet-system fetal bovine serum and 1 μg/ml doxycycline. After 5 min and 72 h, the supernatant was collected, and luciferase activity is measured by QUANTI-Luc reagent (as per manufacturer’s instructions) onto a 96-well plate (Corning) with a microplate reader (Synergy H1 BioTek). Each experiment was performed in triplicate and the results were analyzed with an unpaired t test on Prism 9.3.1 (GraphPad).A549 Dual and A549 Dual KO-RIG-I cells (InvivoGen) were transduced with pInducer CHIKV nsP1 and pInducer CHIKV nsP1-mut lentivirus in appropriate media containing 8 μg/ml polybrene (Sigma-Aldrich). Media was replaced after 24 h and at 48 h selected with geneticin G418 (Gibco) and 1 μg/ml doxycycline was added for induction of protein expression. CHIKV nsP1 and CHIKV nsP1-mut expression was verified by Western blot D. A549 D7GpppAmU were incubated with nsP1-mut under 4°C overnight before preparing Cryo-EM grids. The carbon side of the Quantifoil R1.2/1.3 gold 300 mesh grid was covered with one layer of graphene following a published protocol (SAH and mprotocol . After ghttps://emcore.ucsf.edu/ucsf-software) (https://cryosparc.com/) (The collected movies of different samples were motion-corrected and dose-weighted using MotionCor2 (oftware) . The outrc.com/) . After crc.com/) . The det6Z0V (http://pymol.org). The H37 was manually modeled into the current structure by aligning to template m7GTP-bound WT nsP1 (PDB 7FGG (Deposited nsP1 structure (PDB: 6Z0V ) was use6Z0V . The ref6Z0V  and PyMOPDB 7FGG ).7GpppAmU has been deposited in EM Database under the accession code EMD-36159. The corresponding atomic coordinates have been deposited in the Protein Data Bank under accession code 8JCE.The cryo-EM density map for the CHIKV nsP1(H37A) in complex with SAH and mThe authors declare that they have no conflict interests with the contents of this article.