Biology subjects

Wu, P.

Publications and source records attributed to Wu, P..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Highly multiplexed, image-based pooled screens in primary cells and tissues with PerturbView

Optical pooled screening (OPS) is a highly scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture, due to limitations associated with in situ sequencing (ISS) of perturbation barcodes. Here, we developed PerturbView, an OPS technology that leverages in vitro transcription (IVT) to amplify barcodes prior to ISS, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in iPSC-derived neurons, primary immune cells, and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF{kappa}B signaling. Furthermore, we combined PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in vivo screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

systems biology↗

bioRxiv2023

Power-law growth models explain incidences and sizes of pancreatic cancer precursor lesions and confirm spatial genomic findings

Pancreatic ductal adenocarcinoma is a rare but lethal cancer. Recent evidence reveals that pancreatic intraepithelial neoplasms (PanINs), the microscopic precursor lesions in the pancreatic ducts that can give rise to invasive pancreatic cancer, are significantly larger and more prevalent than previously believed. Better understanding of the growth law dynamics of PanINs may improve our ability to understand how a miniscule fraction of these lesions makes the transition to invasive cancer. Here, using artificial intelligence (AI)-based three-dimensional (3D) tissue mapping method, we measured the volumes of >1,000 PanIN and found that lesion size is distributed according to a power law with a fitted exponent of -1.7 over > 3 orders of magnitude. Our data also suggest that PanIN growth is not very sensitive to the pancreatic microenvironment or an individuals age, family history, and lifestyle, and is rather shaped by general growth behavior. We analyze several models of PanIN growth and fit the predicted size distributions to the observed data. The best fitting models suggest that both intraductal spread of PanIN lesions and fusing of multiple lesions into large, highly branched structures drive PanIN growth patterns. This work lays the groundwork for future mathematical modeling efforts integrating PanIN incidence, morphology, genomic, and transcriptomic features to understand pancreas tumorigenesis, and demonstrates the utility of combining experimental measurement of human tissues with dynamic modeling for understanding cancer tumorigenesis.

biophysics↗

bioRxiv2023

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge, while incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to 7 genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expressions of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/L, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased FAD biosynthesis, enhanced flux entering the TCA cycle and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories. Impact StatementIn this study, yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and successfully incorporating modules for large-scale disulfide bond formation. KmYACs were stably propagated in K. marxianus without compromising the normal growth of the host, irrespective of the selection of telomeres (either Tetrahymena or K. marxianus) and configuration (either linear or circular). KmYACs notably enhanced the expressions of various heterologous proteins, with further yield improvement by supplementing lysine or arginine in the medium. Our findings affirm KmYAC as a robust and versatile platform for transferring large-scale function modules, demonstrating immense potential for both industrial applications and fundamental research.

synthetic biology↗

bioRxiv2023

Targeting CD45 by gene-edited CAR-T cells for leukemia eradication and hematopoietic stem cell transplantation preconditioning

Hematopoietic stem cell transplantation (HSCT) is widely used to treat patients with life-threatening hematologic and immune system disorders. The currently used nontargeted chemo-/radiotherapy conditioning regimens cause tissue injury and induce an array of immediate and delayed adverse effects, which limits the use of this potentially curative treatment. The growing demand to replace canonical conditioning regimens has led to the development of alternative approaches based on antibody-drug conjugates, naked antibodies and CAR T cells. Here, we propose a preconditioning strategy based on targeting CD45 on hematopoietic cells with CAR45 T cells. To avoid fratricide of CD45 CAR T cells, targeted genomic disruption of the CD45 gene was performed in human CD45 CAR T cells in combination with dasatinib treatment. CD45{Delta}CAR45 T cells showed impressive activity in terms of target cell elimination in vitro and depletion of tumor cells in vivo or human hematopoietic cells in humanized immunodeficient mice engrafted with human blood-derived HSCs. CD45{Delta}CAR45 NK cells also exhibited potent killing activity against tumor cell lines and human hematopoietic cells. Therefore, fratricide-resistant CAR45 T and NK cells have the potential to provide the benefits of full myeloablative conditioning and therapy for hematologic malignancies. Thus, we provide the proof of concept for the generation and preclinical efficacy of CAR T cells directed against CD45-expressing cells.

molecular biology↗

bioRxiv2023

An axonemal intron splicing program sustains Plasmodium male development

Differentiation of male gametocyte into flagellated fertile male gamete relies on the axoneme assembly, a major component of male development for mosquito transmission of malaria parasite. RNA-binding protein (RBP)-mediated post-transcription regulation plays important roles in eukaryotic sexual development, including the Plasmodium female development. However, the role of RBP in defining the Plasmodium male transcriptome and its function in the male gametogenesis remain elusive. Here, we screened the gender expression of the genome-wide RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with spliceosome E complex and regulates splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal proteins expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. Therefore, the splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcription regulation essential for Plasmodium male development.

microbiology↗

bioRxiv2023

Guided Diffusion for molecular generation with interaction prompt

Molecular generative models have demonstrated their potential in designing molecules from scratch with high binding affinities in a pre-determined protein pocket and could be combined with traditional structural based drug design strategy. However, the generative processes of such models are random and the atomic interaction information between ligand and protein are ignored. On the other hand, the ligand has high propensity to bind with residues called hotspots. Hotspot residues contribute to the majority of the binding free energies and have been recognized as appealing targets for designed molecules. In this work, we develop an interaction prompt guided diffusion model-InterDiff to deal with the challenges. Four kinds of atomic interactions are involved in our model and represented as learnable vector embeddings. These embeddings serve as conditions for each residue to guide the molecular generative process. Comprehensive in-silico experiments evince that our model could generate molecules with desired ligand-protein interactions in a guidable way. Furthermore, we validate InterDiff on two realistic protein-based therapeutic agents. Results show that InterDiff could generate molecules with better or similar binding mode compared to known targeted drugs.

bioinformatics↗

bioRxiv2023

Distinct 3D contacts and phenotypic consequences of adjacent non-coding loci in the epigenetically quiescent regions

Non-coding regions of the human genome are important for functional regulations, but their mechanisms remain elusive. We used machine learning to guide a CRISPR screening on hubs (i.e. non-coding loci forming many 3D contacts) and significantly increased the discovery rate of hubs essential for cell growth. We found no clear genetic or epigenetic differences between essential and nonessential hubs, but we observed that some neighboring hubs in the linear genome have distinct spatial contacts and opposite effects on cell growth. One such pair in an epigenetically quiescent region showed different impacts on gene expression, chromatin accessibility and chromatin organization. We also found that deleting the essential hub altered the genetic network activity and increased the entropy of chromatin accessibility, more severe than that caused by deletion of the nonessential hub, suggesting that they are critical for maintaining an ordered chromatin structure. Our study reveals new insights into the system-level roles of non-coding regions in the human genome.

genetics↗

bioRxiv2023

The canonical single-stranded DNA binding protein SSB is not an essential replication protein but an RNA chaperon in the hyperthermophilic archaeon Saccharolobus islandicus REY15A

Single-stranded DNA binding proteins (SSBs) have been regarded as indispensable factors in all three domains of life since they play vital roles in DNA replication. Herein, we report that genes coding for the canonical SSB (SisSSB) and the non-canonical SSB (SisDBP) in the hyperthermophilic archaeon Saccharolobus islandicus REY15A can both be deleted. The growth is not affected, and the cell cycle progression and genome stability of the deletion strains is not impaired, suggesting that SisSSB and SisDBP are not essential for cell viability. Interestingly, at a lower temperature (55{degrees}C), the protein level of SisSSB increases [~]1.8 fold in the wild type and the growth of {Delta}Sisssb and {Delta}Sisssb{Delta}Sisdbp is retarded. SisSSB exhibits melting activity on dsRNA and DNA/RNA hybrid in vitro and unwinding RNA hairpin in Escherichia coli. Furthermore, the core SisSSB domain is able to complement the absence of the cold shock proteins CspABGE in E. coli, suggesting that SisSSB functions as RNA chaperon. We show that a two-fold overexpression of SisSSB is beneficial to the cell growth at lower temperature, but it has detrimental effect on the cell growth and cell cycle progression at normal growth temperature, which differs from bacterial Csp proteins. Importantly, these in vitro and in vivo activities are conserved in SSB subtype SSB-1 in Crenarchaeota species that lack bacterial Csp homologs. Overall, we have clarified the function of the archaeal canonical SSB which does not function as a DNA processing factor, but plays a role in processes requiring dsRNA or DNA/RNA hybrid unwinding.

microbiology↗

bioRxiv2023

A Series of Composited Tumor DNA Reference Materials Containing Three Genes and Ten Mutation Positions for CNV and SNV Detection

Processes in clinic for tumors diagnosis and treatment need reference materials (RMs) to evaluate and calibrate. However, no RMs can provides properties of copy number variation (CNV) and single nucleotide variants (SNV) of genes EGFR, HER2, MET, PIK3CA, KRAS, BRAF, NRAS simultaneously. In this study, we used commercial cell lines to construct a series of tumor RMs containing property mentioned above. Furthermore, we evaluated their stability, homogeneity, and commutability by droplet digital PCR and next generation sequencing technology. The results showed that, for tumor CNV gDNA RM, the copy number is 7.3 copies/L (EGFR), 5.3 copies/L (HER2) and 8.2 copies/L (MET). For tumor 5% SNV gDNA RM, the mutation frequency of each mutation position showed as follow: EGFR-E746A750 (24.6%), EGFR-L858R (5.8%), EGFR-T790M (5.5%), EGFR-G719S (6.6%), PIK3CA-E545K (4.7%), PIK3CA-H1047R (5.8%), KRAS-G13D (8.2%), KRAS-G12D (6.5%), BRAF-V600E (4.6%), NRAS-Q61K (8.5%). All variable coefficient (CV) of tumor gDNA RM for homogeneity were less than 7%, that of CNV+SNV ctDNA RM were less than 17%. Besides, the CV for commutability of the all types of RMs were less than 17%. These RMs can be applied into a wide range type of sequencing panels and provides a closer simple background.

molecular biology↗

bioRxiv2023

Gap junctions in Turing-type periodic feather pattern formation

Periodic patterning requires coordinated cell-cell interactions at the tissue level. Turing showed, using mathematical modeling, how spatial patterns could arise from the reactions of a diffusive activator-inhibitor pair in an initially homogenous two-dimensional field. Most activators and inhibitors studied in biological systems are proteins, and the roles of cell-cell interaction, ions, bioelectricity, etc. are only now being identified. Gap junctions (GJs) mediate direct exchanges of ions or small molecules between cells, enabling rapid long-distance communications in a cell collective. They are therefore good candidates for propagating non-protein-based patterning signals that may act according to the Turing principles. Here, we explore the possible roles of GJs in Turing-type patterning using feather pattern formation as a model. We found seven of the twelve investigated GJ isoforms are highly dynamically expressed in the developing chicken skin. In ovo functional perturbations of the GJ isoform, connexin 30, by siRNA and the dominant-negative mutant applied before placode development led to disrupted primary feather bud formation, including patches of smooth skin and buds of irregular sizes. Later, after the primary feather arrays were laid out, inhibition of gap junctional intercellular communication in the ex vivo skin explant culture allowed the emergence of new feather buds in temporal waves at specific spatial locations relative to the existing primary buds. The results suggest that gap junctional communication may facilitate the propagation of long-distance inhibitory signals. Thus, the removal of GJ activity would enable the emergence of new feather buds if the local environment is competent and the threshold to form buds is reached. We propose Turing-based computational simulations that can predict the appearance of these ectopic bud waves. Our models demonstrate how a Turing activator-inhibitor system can continue to generate patterns in the competent morphogenetic field when the level of intercellular communication at the tissue scale is modulated.

developmental biology↗

bioRxiv2023

Ser14-RPN6 Phosphorylation Mediates the Activation of 26S Proteasomes by cAMP and Protects against Cardiac Proteotoxic Stress in Mice

BackgroundA better understanding of the regulation of proteasome activities can facilitate the search for new therapeutic strategies. A cell culture study shows that cAMP-dependent protein kinase (PKA) activates the 26S proteasome by phosphorylating Ser14 of RPN6 (pS14-RPN6), but this discovery and its physiological significance remain to be established in vivo. MethodsMale and female mice with Ser14 of Rpn6 mutated to Ala (S14A) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created and used along with cells derived from them. cAMP/PKA were manipulated pharmacologically. Ubiquitin-proteasome system (UPS) functioning was evaluated with the GFPdgn reporter mouse and proteasomal activity assays. Impact of S14A and S14D on proteotoxicity was tested in mice and cardiomyocytes overexpressing the misfolded protein R120G-CryAB (R120G). ResultsPKA activation increased pS14-Rpn6 and 26S proteasome activities in wild-type (WT) but not S14A embryonic fibroblasts (MEFs), adult cardiomyocytes (AMCMs), and mouse hearts. Basal 26S proteasome activities were significantly greater in S14D myocardium and AMCMs than in WT counterparts. S14D::GFPdgn mice displayed significantly lower myocardial GFPdgn protein but not mRNA levels than GFPdgn mice. In R120G mice, a classic model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with non- transgenic littermates, which was not always associated with reduction of other phosphorylated PKA substrates. Cultured S14D neonatal cardiomyocytes displayed significantly faster proteasomal degradation of R120G than WT neonatal cardiomyocytes. Compared with R120G mice, S14D/S14D::R120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates and of aberrant CryAB protein aggregates, less reactivation of fetal genes and cardiac hypertrophy, and delays in cardiac malfunction. ConclusionsThis study establishes in animals that pS14-Rpn6 mediates the activation of 26S proteasomes by PKA and that the reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifies a new therapeutic target to reduce cardiac proteotoxicity.

cell biology↗

bioRxiv2023

DNA-PK and the TRF2 iDDR inhibit MRN-initiated resection at leading-end telomeres

Telomeres replicated by leading-strand synthesis lack the 3 overhang required for telomere protection. Surprisingly, resection of these blunt telomere is initiated by the telomere-specific 5 exonuclease Apollo rather than the Mre11-Rad50-Nbs1 (MRN) complex, the nuclease that acts at DNA breaks. Without Apollo, leading-end telomeres undergo fusion, which, as demonstrated here, are mediated by alternative End Joining. Here, we show that DNA-PK and TRF2 coordinate the repression of MRN at blunt telomeres. DNA-PK represses an MRN-dependent long range resection at blunt telomeres, while the endonuclease activity of MRN/CtIP, which could cleave DNA-PK off of blunt telomere ends, is inhibited in vitro and in vivo by the iDDR of TRF2. AlphaFold-Multimer predicts a conserved association of the iDDR with Rad50 potentially interfering with CtIP binding and MRN endonuclease activation. We propose that repression of MRN-mediated resection is a conserved aspect of telomere maintenance and represents an ancient feature of DNA-PK and the iDDR.

molecular biology↗

bioRxiv2023

Transient EZH2 suppression by Tazemetostat during in vitro expansion maintains T cell stemness and improves adoptive T cell therapy

The histone methyltransferase enhancer of zeste homolog 2 (EZH2)-mediated epigenetic regulation of T cell differentiation in acute infection has been extensively investigated. However, the role of EZH2 in T cell exhaustion remains under-explored. Here, using in vitro exhaustion models, we demonstrated that transient inhibition of EZH2 in T cells before the phenotypic onset of exhaustion with a clinically approved inhibitor, Tazemetastat, delayed their dysfunctional progression and maintained T cell stemness and polyfunctionality while having no negative impact on cell proliferation. Tazemetestat induced T cell epigenetic reprogramming and increased the expression of the self-renewing T cell transcription factor TCF1 by reducing its promoter H3K27 methylation preferentially in rapidly dividing T cells. In a murine melanoma model, T cells pre-treated with tazemetastat exhibited a superior response to anti-PD-1 blockade therapy after adoptive transfer. Collectively, these data unveil the potential of transient epigenetic reprogramming as a potential intervention to be combined with checkpoint blockade for immune therapy.

immunology↗

bioRxiv2023

DYRK1A interacts with Tuberous Sclerosis complex and promotes mTORC1 activity

DYRK1A, a ubiquitously expressed kinase, is linked to the dominant intellectual developmental disorder, microcephaly and Down syndrome in humans. It regulates numerous cellular processes such as cell cycle, vesicle trafficking and microtubule assembly. DYRK1A is a critical regulator of organ growth; however, how it regulates organ growth is not fully understood. Here, we show that the knockdown of DYRK1A results in reduced cell size, which depends on mTORC1. Using proteomic approaches, we found that DYRK1A interacts with the Tuberous sclerosis complex (TSC) proteins, namely TSC1 and TSC2, which negatively regulate mTORC1 activation. Further, we show that DYRK1A phosphorylates TSC2 at T1462, a modification known to inhibit TSC activity and promote mTORC1 activity. We also found that the reduced cell growth upon knockdown of DYRK1A can be rescued by overexpression of RHEB, an activator of mTORC1. Our findings suggest that DYRK1A inhibits TSC complex activity through inhibitory phosphorylation on TSC2, thereby promoting mTORC1 activity. Further, using the Drosophila neuromuscular junction as a model, we show that the mnb, the fly homologues of DYRK1A, is rescued by RHEB overexpression, suggesting a conserved role of DYRK1A in TORC1 regulation.

molecular biology↗

bioRxiv2023

Sex-dependent Differences in the Genomic Profile of Lingual Sensory Neurons in Naive and Tongue-Tumor Bearing Mice

Mechanisms of sex-dependent orofacial pain are widely understudied. A significant gap in knowledge exists about comprehensive regulation of tissue-specific trigeminal sensory neurons in diseased state of both sexes. Using RNA sequencing of FACS sorted retro-labeled sensory neurons innervating tongue tissue, we determined changes in transcriptomic profiles in males and female mice under naive as well as tongue-tumor bearing conditions Our data revealed the following interesting findings: 1) Tongue tissue of female mice was innervated with higher number of trigeminal neurons compared to males; 2) Naive female neurons innervating the tongue exclusively expressed immune cell markers such as Csf1R, C1qa and others, that werent expressed in males. This was validated by Immunohistochemistry. 4) Accordingly, immune cell markers such as Csf1 exclusively sensitized TRPV1 responses in female TG neurons. 3) Male neurons were more tightly regulated than female neurons upon tumor growth and very few differentially expressed genes (DEGs) overlapped between the sexes, 5) Male DEGs contained higher number of transcription factors whereas female DEGs contained higher number of enzymes, cytokines and chemokines. Collectively, this is the first study to characterize the effect of sex as well as of tongue-tumor on global gene expression, pathways and molecular function of tongue-innervating sensory neurons.

neuroscience↗

bioRxiv2022

Nanobodies for the treatment of SARS-CoV-2 in animals: a meta-analysis

This meta-analysis aimed to find the effect of variable domain of heavy-chain antibodies (VHHs) for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in animals. The databases of the PubMed, China National Knowledge Infrastructure (CNKI), Wan fang data, Cochrane Library, and Embase were searched for articles published before August 2022 on the protective effects of VHHs in animals. The articles retrieved were screened using inclusion and exclusion criteria. The data were analyzed using Review Manager 5.4. Six articles were selected from 667 articles based on the inclusion and exclusion criteria in VHHs. A forest plot showed that VHHs could offer protection against SARS-CoV-2 infection in animals [Mantel-Haenszel (MH) = 172.94, 95% confidence interval (CI) = (43.96, 678.42), P < 0.00001]. There was almost no heterogeneity in this study (I2 = 0). A funnel plot showed that the bias of the data analysis was small. This is a special meta-analysis proved that VHHs could treat and prevent SARS-CoV-2 in animals.

immunology↗

bioRxiv2022

Chemoenzymatic Measurement of Cell-surface Glycan in Single-cell Multiomics: LacNAc as an Example

Despite the rich information of a cells physiological state encoded in the dynamic changes of cell-surface glycans, methods of capturing glycosylation states at the single-cell level are quite limited. Here we report a chemoenzymatic single-cell N-acetyllactosamine (LacNAc) detection method via tagging the LacNAc with a specific DNA barcode. Compared to the lectin-based glycan detection, the chemoenzymatic labeling does not change the transcriptional status of immune cells and is more compatible with scRNA-seq. Integrated analysis of LacNAc and transcriptome of T cells at a single-cell level reveals that the quantity of cell-surface LacNAc is significantly upregulated in activated CD8+ T cells but maintained at the basal level in quiescent CD8+ T cells (i.e., naive and central memory T cells). Further analysis confirms that the LacNAc level is positively correlated to the glycolytic activity of CD8+ T cells at all statues. Taken together, our study demonstrates the feasibility of chemoenzymatic detection of cell-surface glycan in single-cell RNA sequencing-based multiomics with information of TCR sequence and cell-surface epitopes (i.e., scTCR and CITE-seq) and offers a new way to characterize the biological role of glycan in diversified physiological states.

immunology↗

bioRxiv2022

An evolutionally conserved archaeal KEOPS complex functions in DNA repair

KEOPS is a multi-subunit protein complex conserved in eukaryotes and archaea. It is composed of Pcc1, Kae1, Bud32, Cgi121, and Gon7 in eukaryotes and is primarily involved in N6-threonylcarbamoyl adenosine (t6A) modification of tRNAs. Recently, KEOPS is reported to participate in homologous recombination repair in yeast. To characterize the KEOPS in archaea (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus. We show that aKEOPS also possesses five subunits, Pcc1, Kae1, Bud32, Cgi121, and Pcc1-like (or Gon7-like), just as eukaryotic KEOPS. Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1-Pcc1-Pcc1-Kae1), suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1-like, can be deleted in the wild type and in a t6A modification complementary strain named TsaKI, implying that aKEOPS complex is essential for an additional cellular process in this archaeon. Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type which is partially rescued in TsaKI. These results suggest that aKEOPS plays an essential role independent of cellular t6A modification level. In addition, archaeal Cgi121 possesses dsDNA-binding activity which relies on its tRNA 3 CCA tail binding module. Our study clarifies the subunit organization of archaeal KEOPS and suggests of an origin of eukaryotic Gon7. The study also reveals a possible link between the function in t6A modification and the additional function presumably homologous recombination.

microbiology↗