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Zhao, Z.

Publications and source records attributed to Zhao, Z..

At least 397 records · Page 22Linked to original sources
bioRxiv2021

QSAR Model Based Gradient Boosting Regression of N-Arylsulfonyl-Indole-2-Carboxamide Derivatives as Inhibitors for Fructose-1,6-Bisphosphatase

As is known to all, diabetes metellius is a global health threaten and it has caused worldwide attention of scientists. To get a better investigation of the drug design of diabetes, we used heuristic method to established the linear model and used Gradient Boosting Regression to establish the nonlinear model of Fructose-1,6-Bisphosphatse inhibitor successively. In this study, 84 derivatives of N-Arylsulfonyl-Indole-2-Carboxamide were introduced into the models, two outstanding QSAR models with 2 molecule descriptors were established successfully. Grandient Boosting Regression rendered a good correlation with R2 of 0.943 and MSE of 0.135 for the training set, 0.916 and 0.213 for test set, which also proves the feasibility of the implementation of the new method GBR in the field of QSAR. Meanwhile, the optimal model displayed wonderful statistical significance. This study shows unlimited potential for design of new drugs for diabetes.

biochemistry↗
bioRxiv2021

Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery

Neuronal extracellular vesicles (EVs) can be locally released from presynaptic terminals, carrying cargoes that are important in intercellular signaling and disease. EVs are derived from endosomes, but it remains unclear how synaptic cargoes are directed to the EV pathway, rather than undergoing conventional retrograde endosomal transport and degradation. Here, we find that the clathrin-mediated endocytic machinery plays an unexpected role in maintaining a release-competent pool of synaptic EV cargoes. Endocytic mutants, including nervous wreck (nwk), Shibire/Dynamin, and AP-2, exhibit local depletion of multiple cargoes in EV donor terminals. Accordingly, nwk mutants phenocopy synaptic plasticity defects associated with loss of the EV cargo Synaptotagmin-4, and suppress lethality upon overexpression of the EV cargo Amyloid Precursor Protein. These EV defects are genetically separable from canonical functions of endocytic proteins in synaptic vesicle recycling and synaptic growth. This endocytic pathway opposes the endosomal retromer complex to regulate EV cargo levels, and acts upstream of synaptic cargo removal by retrograde axonal transport. Our data suggest a novel molecular mechanism that protects EV cargoes from local depletion at synapses.

cell biology↗
bioRxiv2021

Super-resolution imaging of highly curved membrane structures in giant vesicles encapsulating molecular condensates

Molecular crowding is an inherent feature of the cell interior. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (polyethylene-glycol and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs loaded with macromolecules exhibit highly curved structures such as internal nanotubes. In addition, upon liquid-liquid phase separation as inside living cells, the membrane of GUVs encapsulating an aqueous two-phase system deforms to form apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes and kinks, have dimensions below optical resolution and if resolved, can provide information about material properties such as membrane spontaneous curvature and intrinsic contact angle describing the wettability contrast of the encapsulated phases to the membrane. Previous experimental studies were based on conventional optical microscopy which cannot resolve these membrane and wetting properties. Here, we studied these structures with super-resolution microscopy, namely stimulated emission depletion (STED) microscopy, together with microfluidic manipulation. We demonstrate the cylindrical nature of the nanotubes with unprecedented detail based on the superior resolution of STED and automated data analysis. The spontaneous curvature deduced from the nanotube diameters is in excellent agreement with theoretical predictions. Furthermore, we were able to resolve the membrane "kink" structure as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle. We find very good agreement between the directly measured values and the theoretically predicted ones based on the apparent contact angles on the micrometer scale. During different stages of cellular events, biomembranes undergo a variety of shape transformations such as the formation of buds and nanotubes regulated by membrane necks. We demonstrate that these highly curved membrane structures are amenable to STED imaging and show that such studies provide important insights in the membrane properties and interactions underlying cellular activities.

biophysics↗
bioRxiv2021

A pore-forming protein drives macropinocytosis to facilitate toad water maintaining

Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a secretory aerolysin family pore-forming protein and trefoil factor complex {beta}{gamma}-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for {beta}{gamma}-CAT in toad water maintaining. Deletion of toad skin secretions, in which {beta}{gamma}-CAT is a major component, increased animal mortality under hypertonic stress. {beta}{gamma}-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, {beta}{gamma}-CAT stimulated macropinocytosis to facilitate water intake and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporins, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress.

cell biology↗
bioRxiv2021

Critical Assessment of Metagenome Interpretation - the second round of challenges

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the community-driven initiative for the Critical Assessment of Metagenome Interpretation (CAMI). In its second challenge, CAMI engaged the community to assess their methods on realistic and complex metagenomic datasets with long and short reads, created from [~]1,700 novel and known microbial genomes, as well as [~]600 novel plasmids and viruses. Altogether 5,002 results by 76 program versions were analyzed, representing a 22x increase in results. Substantial improvements were seen in metagenome assembly, some due to using long-read data. The presence of related strains still was challenging for assembly and genome binning, as was assembly quality for the latter. Taxon profilers demonstrated a marked maturation, with taxon profilers and binners excelling at higher bacterial taxonomic ranks, but underperforming for viruses and archaea. Assessment of clinical pathogen detection techniques revealed a need to improve reproducibility. Analysis of program runtimes and memory usage identified highly efficient programs, including some top performers with other metrics. The CAMI II results identify current challenges, but also guide researchers in selecting methods for specific analyses.

bioinformatics↗
bioRxiv2021

Atp13a5 Marker Reveals Pericytes of The Central Nervous System in Mice

Perivascular mural cells including vascular smooth cells (VSMCs) and pericytes are integral components of the vascular system. In the central nervous system (CNS), pericytes are also known as the guardian of the blood-brain barrier, blood-spinal cord barrier and blood-retinal barrier, and play key roles in maintaining cerebrovascular and neuronal functions. However, the functional difference between CNS and peripheral pericytes has not been resolved at the genetic and molecular levels. Hence, the generation of reliable CNS pericyte-specific models and genetic tools remains very challenging. Here, we report a new CNS pericyte marker in mice. This cation-transporting ATPase 13A5 (Atp13a5) marker is highly specific to the pericytes in brain, spinal cord and retina. We generated a transgenic model with a knock-in tdTomato reporter and Cre recombinase. The tdTomato reporter reliably labels the CNS pericytes, but not found in any other CNS cell types including closely related VSMCs, or in peripheral organs. More importantly, Atp13a5 is turned on at embryonic day E15, suggesting brain pericytes are shaped by the developing neural environment. We hope that the new tools will allow us to further explore the heterogeneity of pericytes and achieve a better understanding of CNS pericytes in health and diseases.

neuroscience↗
bioRxiv2021

Adenylate Kinase 2 deficiency causes NAD+ depletion and impaired purine metabolism during myelopoiesis

Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency that affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, lymphopenia, and deafness. The disease is caused by biallelic loss of function in mitochondrial Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of AMP to ADP, as substrate for ATP synthesis. Accordingly, declining oxidative phosphorylation has been postulated as the driver of disease pathology. The mechanistic basis, however, remains incompletely understood. Single cell RNA-sequencing of patient bone marrow cells implicated altered RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings, we developed a disease model based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. We found that AK2-deficient myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels of the purine nucleotide precursor IMP, decreased cellular RNA content, ribosome subunit expression, protein synthesis and a profoundly hypo-proliferative phenotype. The rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the disease phenotype, pointing to AMP catabolism as a possible metabolic adaptation to mitigate AMP-mediated toxicity. Inducing an adenosine disequilibrium in control cells produced a similar myeloid maturation defect. This study shows that AK2 deficiency globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism. AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity may contribute to the failure of myelopoiesis in Reticular Dysgenesis.

cell biology↗
bioRxiv2021

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV 1/SIVcpz lentiviral lineage

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy - termed the "R-clamp" - that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution. AUTHOR SUMMARYViral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

microbiology↗
bioRxiv2021

Regulation Network of Colorectal Cancer Specific Enhancers in Progression of Colorectal Cancer

Enhancers regulate multiple genes through higher-order chromatin structure and further affect cancer progression. Epigenetic changes in cancer cells activate several cancer specific enhancers that are silenced in normal cells. These cancer specific enhancers are potential therapeutic targets of cancer. However, functions and regulation network of colorectal cancer specific enhancers are still unknown. Here in this study, we profile colorectal cancer specific enhancers and reveal the regulation network of these enhancers by analysis of HiChIP, Hi-C and RNA-seq data. We propose the regulation network of colorectal cancer specific enhancers plays important role in progression of colorectal cancer.

genetics↗
bioRxiv2021

Landscape of microenvironment in Randall's plaque by single-cell sequencing

Randalls plaque is significantly associated with the occurrence of nephrolithiasis. However, the microenvironment of Randalls plaque is poorly characterized. To investigate the microenvironment of Randalls plaque, we analyzed single-cell RNA data of 3 Randalls plaque and 3 normal renal papillae tissue and identified 11 different cell types. We screened differentially expressed genes among all cell types between Randalls plaque and normal renal papillae. The microenvironment showed two cell types with multiple stone formation-associated transcriptomic programs. Contrary to previous studies, we did not observe macrophage M1/M2 imbalance. Notably, we detected ossification-associated macrophage is enriched in Randalls plaque and validated GPNMB and ACP5 were potential biomarkers on the ossification-associated macrophage. We also identified an endothelial subset harboring active communication (COL15A1+ PCDH17+ endothelial, DPECs) with other cells. Together with Immunofluorescence, we validated ossification-associated macrophage and DPECs are enriched in Randalls plaque tissue. Finally, cell-to-cell communication revealed that Loop of Henle, DPECs, and osteoblasts-associated macrophages was the main source of SPP1 signaling. Our work will further the understanding of the microenvironment among Randalls plaque tissues and provide deep insight into immune modulation.

bioinformatics↗
bioRxiv2021

Rapid Electronic Diagnostics of Ebola Virus with Synthetic Nanobody-Conjugated Gold Nanoparticles

Successful control of emerging infectious diseases requires accelerated development of fast, affordable, and accessible assays to be widely implemented at a high frequency. Here we present a generalizable assay platform, nanobody-functionalized nanoparticles for rapid, electronic detection (Nano2RED), demonstrated in the detection of Ebola and COVID-19 antigens. To efficiently generate high-quality affinity reagents, synthetic nanobody co-binders and mono-binders with high affinity, specificity, and stability were selected by phage display screening of a vastly diverse, rationally randomized combinatorial library, bacterially expressed and site-specifically conjugated to gold nanoparticles (AuNPs) as multivalent in-solution sensors. Without requiring fluorescent labelling, washing, or enzymatic amplification, these AuNPs reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or simple electronic circuitry. With nanobodies against an Ebola virus secreted glycoprotein (sGP) and a SARS-CoV-2 spike protein receptor binding domain (RBD) as targets, Nano2RED showed a high sensitivity (limit of detection of [~]10 pg/mL for sGP and [~]40 pg/mL for RBD in diluted human serum), a high specificity, and a large dynamic range ([~]7 logs). Unlike conventional assays where slow mass transport for surface binding limits the assay time, Nano2RED features fast antigen diffusion at micrometer scale, and can be accelerated to deliver results within a few minutes. The rapid detection, low material cost (estimated < $0.01 per test), inexpensive and portable readout system (< $5 and < 100 cm3), and digital data output, make Nano2RED particularly suitable for screening of patient samples with simplified operation and accelerated data transmission. Our method is widely applicable for prototyping diagnostic assays for other antigens from new emerging viruses.

bioengineering↗
bioRxiv2021

Cell-type specific requirement for pyruvate dehydrogenase in hematopoiesis and leukemia development.

Cancer cells have different metabolic requirements as compared to their corresponding normal tissues. This is thought to reflect metabolic reprogramming during transformation. An alternative possibility is that some metabolic requirements of cancer cells reflect a maintenance of the metabolism of the specific normal cell type from which cancer cells originate. Here, we investigate this hypothesis by comparing glucose use in normal hematopoiesis and in leukemia. T cell progenitors in the thymus were glucose avid and oxidized more glucose in the tricarboxylic acid (TCA) cycle through pyruvate dehydrogenase (PDH) as compared to hematopoietic stem cells (HSCs) or other hematopoietic cells. PDH deletion reduced the number of double positive (DP) T cell progenitors but had no effect on HSCs, myeloid progenitors and other hematopoietic cells we examined. PDH deletion blocked the development of T cell leukemia from Pten-deficient DP progenitors, but not the development of a myeloid neoplasm from Pten-deficient HSCs or myeloid progenitors. Therefore, the requirement of glucose oxidation for leukemia development is inherited from the normal cell of origin and occurs independently of the driver genetic lesion. PDH was not required in vivo to generate acetyl-CoA or maintain levels of TCA cycle metabolites but to prevent pyruvate accumulation and to maintain glutathione levels and redox homeostasis.

cancer biology↗
bioRxiv2021

Genome sequencing and functional genes comparison between Sphingopyxis USTB-05 and Sphingomonas morindae NBD5

Sphingomonadaceae has a large number of strains that can biodegrade hepatotoxins or environmental pollutants. The latest research reported that certain strains can also produce lutein. Based on the third-generation sequencing technology, we analyzed the whole genome sequence and compared related functional genes of two strains of Sphingomonadaceae isolated from different habitats. The genome of Sphingopyxis USTB-05 was 4,679,489 bp and contained 4312 protein coding genes. The 4,239,716 bp nuclear genome of Sphingomonas morindae NBD5, harboring 3882 protein coding genes, has two sets of chromosomes. Both strains had lutein synthesis metabolism pathway sharing identical synthetic genes of crtB, crtE, crtI, crtQ, crtL, crtR, atoB, dxs, dxr, ispD, ispE, ispDF, gcpE, ispG, ispH, ispA, ispB and ispU. Sphingopyxis USTB-05 had hepatotoxins microcystins and nodularin metabolic pathways related to 16 genes (ald, ansA, gdhA, crnA, phy, ocd, hypdh, spuC, nspC, speE, murI, murD, murC, hmgL, bioA and glsA), while these genes were not found in Sphingomonas morindae NBD5. The unique protein sequences of strain NBD5 and strain USTB-05 were 155 and 199, respectively. The analysis of whole genome of the two Sphingomonadaceae strains provides insights into prokaryote evolution, the new pathway for lutein production and the new genes for environmental pollutant biodegradation. IMPORTANCEUnderstanding the functional genes related to the special functions of strains is essential for humans to utilize microbial resources. The ability of Sphingopyxis USTB-05 to degrade hepatotoxins microcystins and nodularin has been studied in depth, however the complete metabolic process still needs further elucidation. Sphingomonas morindae NBD5 can produce lutein, and it is necessary to determine whether there is a new pathway of lutein. In this study, the whole genome sequencing of Sphingopyxis USTB-05 and Sphingomonas morindae NBD5 were performed for the first time. Lutein synthesis metabolic pathways and synthetic genes were discovered in Sphingomonadaceae. We predicted the existence of new lutein synthesis pathways and revealed most of the genes of the new synthesis pathways. A comparative analysis of the functional genes of the two strains revealed that Sphingopyxis USTB-05 contains a large number of functional genes related to the biodegradation of hepatotoxins or hexachlorocyclohexane. Among them, the functional genes related to the biodegradation and metabolism of hexachlorocyclohexane had not been previously reported. These findings lay the foundation for the biosynthesis of lutein using Sphingomonas morindae NBD5 or Sphingopyxis USTB-05 and the application of Sphingopyxis USTB-05 for the biodegradation of hepatotoxins microcystins and nodularin or environmental pollutants.

microbiology↗
bioRxiv2021

Antibody responses to SARS-CoV-2 mRNA vaccines are detectable in saliva

Vaccines are critical for curtailing the COVID-19 pandemic (1, 2). In the USA, two highly protective mRNA vaccines are available: BNT162b2 from Pfizer/BioNTech and mRNA-1273 from Moderna (3, 4). These vaccines induce antibodies to the SARS-CoV-2 S-protein, including neutralizing antibodies (NAbs) predominantly directed against the Receptor Binding Domain (RBD) (1-4). Serum NAbs are induced at modest levels within [~]1 week of the first dose, but their titers are strongly boosted by a second dose at 3 (BNT162b2) or 4 weeks (mRNA-1273) (3, 4). SARS-CoV-2 is most commonly transmitted nasally or orally and infects cells in the mucosae of the respiratory and to some extent also the gastrointestinal tract (5). Although serum NAbs may be a correlate of protection against COVID-19, mucosal antibodies might directly prevent or limit virus acquisition by the nasal, oral and conjunctival routes (5). Whether the mRNA vaccines induce mucosal immunity has not been studied. Here, we report that antibodies to the S-protein and its RBD are present in saliva samples from mRNA-vaccinated healthcare workers (HCW). Within 1-2 weeks after their second dose, 37/37 and 8/8 recipients of the Pfizer and Moderna vaccines, respectively, had S-protein IgG antibodies in their saliva, while IgA was detected in a substantial proportion. These observations may be relevant to vaccine-mediated protection from SARS-CoV-2 infection and disease.

microbiology↗
bioRxiv2021

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine Protein Phosphatase 2 (PP2A) holoenzyme, PP2A/B55, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55 recruits p107, a pRB-related tumor suppressor and B55 substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an "HxRVxxV619-625" short linear motif (SLiM) in p107 as necessary for B55 binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, "p[ST]-P-x(5-10)-[RK]-V-x-x-[VI]-R". Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55 groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55 mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55[<] role in multiple cellular processes.

biochemistry↗
bioRxiv2021

Dopamine receptor DOP-1 engages a sleep pathway to modulate swimming in C. elegans

Animals require robust yet flexible programs to support locomotion. While it is clear that a variety of processes must be engaged to ensure rhythmic actions, the exact mechanisms remain largely unknown. Here we report a novel pathway that connects the D1-like dopamine receptor DOP-1 with a sleep mechanism to modulate swimming in C. elegans. We show that DOP-1 plays a negative role in sustaining swimming behavior. By contrast, a pathway through the D2-like dopamine receptor DOP-3 negatively regulates the initiation of swimming, but its impact fades quickly over a few minutes. We find that DOP-1 and the GPCR kinase GRK-2 function in the sleep interneuron RIS, where DOP-1 modulates the secretion of a sleep neuropeptide FLP-11. Our genetic studies further show that DOP-1 and FLP-11 act in the same pathway to modulate swimming. Together, these results delineate a functional connection between a dopamine receptor and a sleep program to regulate swimming in C. elegans. The temporal transition between DOP-3 and DOP-1 pathways highlights the dynamic nature of neuromodulation for rhythmic movements that persist over time. HIGHLIGHTSO_LIThe D1-like dopamine receptor DOP-1 regulates swimming at 10 minutes C_LIO_LIAn integrated function of DOP-1 and DOP-3 is required for the continuity of swimming C_LIO_LIDOP-1 and GRK-2 act in the sleep neuron RIS C_LIO_LIFLP-11, a neuropeptide that promotes sleep, negatively regulates swimming C_LI IN BRIEFXu et al. investigated genetic programs that modulate swimming behavior in the nematode C. elegans. They identified a functional link that couples a D1-like dopamine receptor to a sleep program that modulates the sustained phase rather than the initial phase of swimming.

genetics↗
bioRxiv2021

Genomic architecture of 5S rDNA cluster and its variations within and between species

Ribosomal genes (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosome. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. Here, we generate ultra-long Nanopore and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform manual assembly of the cluster using the long reads that carry these units, which led to an assembly of rDNA cluster consisting of up to 167 5S rDNA units. The ordering and copy number of various rDNA units are indicative of separation time between strains. Surprisingly, we observed a drastically lower level of variation in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than C. elegans N2 strain, suggesting a unique mechanism in maintaining the rDNA cluster stability in the N2. Single-copy transgenes landed into the rDNA cluster shows the expected expression in the soma, supporting that rDNA genomic environment is transcriptionally compatible with RNA polymerase II. Delineating the structure and variation of rDNA cluster paves the way for its functional and evolutionary studies.

genomics↗