Alternative Assembly Research Articles

Linkage of alternative exon assembly in Drosophila TrpA1 transcripts

Drosophila TrpA1 (transient receptor potential ankyrin 1) transcripts are alternatively spliced at two distinct sites each with a choice of mutually exclusive exons. The first site determines exon1 encoding the amino terminus to produce either nucleophile-, electrophile- and noxious temperature-gated TRPA1(A) or electrophile- and innocuous warmth-gated TRPA1(B). The second site selects for exon10, resulting in TrpA1 variants with either exon10a or exon10b encoding a domain between the N-terminal ankyrin repeats and the transmembrane segments. Although unbiased assembly would generate TRPA1 with four different domain combinations, the functional impact of these alternative domains remains to be thoroughly examined. Here, we find that there is a relatively strong linkage in mRNA splicing between the two sites in case of TrpA1(B), but not TrpA1(A), transcripts. Our semi-quantitative assay, consisting of reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing, revealed that exon10b is little coupled with TrpA1(B) transcripts, suggesting that only three isoforms, TRPA1(A)-exon10a [denoted as TRPA1(A)], TRPA1(A)-exon10b [TRPA1(A)10b] and TRPA1(B)-exon10a [TRPA1(B)], are present at detectable levels using our method. Interestingly, heterologously expressed TRPA1(A)10b showed elevated sensitivity to low concentrations of N-methyl maleimide (NMM), a cysteine-modifying electrophile, compared with other isoforms. Equivalent isoforms in malaria-transmitting Anopheles gambiae displayed a similar pattern of isoform-dependent NMM dose dependences, suggesting that the chemosensory regulation by selective domain assembly is conserved in insect TRPA1s. Thus, alternative RNA splicing of exon10 is coordinated in conjunction with the first exons, regulating chemical sensitivity of insect TRPA1s.

Adjusting the Number of Functional Groups in Vicinal Bis(trichlorosilylated) Benzenes.

Organo(chloro)silanes are essential chemicals, but the synthesis of compounds of the formula RnSiCl4‒n with defined values of n is usually laborious. Herein, we first disclose that a [4+2]-cycloaddition between readily available Cl3SiC≡CSiCl3 and selected dienes provides facile access to vicinal bis(trichlorosilylated) benzenes and bicyclo[2.2.2]octa-2,5-dienes. Second, we show how the number of six Si‒Cl bonds in 1,2-(Cl3Si)2C6H2Me2 (1) can be selectively reduced to four or two by reaction with pinacol (H2pin) or catechol (H2cat), which act as Si···Si'-bridging and/or Si-chelating ligands. Third, we demonstrate that the thermolysis of compound 6, which contains a benzannulated eight-membered C2(SiCl2)2(μ-pin) ring with Si···Si'-bridging pin moiety, furnishes 13 with a benzannulated five-membered C2(SiCl2)2O ring via the release of pinacolone. Such cyclic disiloxanes are valuable building blocks for specialty silicones; an alternative assembly of the Si‒O‒Si unit by controlled hydrolysis of 1 is not feasible if the four valuable Si‒Cl functionalities are to be retained for further derivatization. Substitution of one Cl atom per Si site of 13 gives the rac-1,3-disila-2-oxaindane rac-15 with high selectivity.

Whole-genome sequencing and identification of antimicrobial peptide coding genes in parsley (Petroselinum crispum), an important culinary and medicinal Apiaceae species.

Parsley is a commonly cultivated Apiaceae species of culinary and medicinal importance. Parsley has several recognized health benefits and the species has been utilized in traditional medicine since ancient times. Although parsley is among the most commonly cultivated members of Apiaceae, no systematic genomic research has been conducted on parsley. In the present work, parsley genome was sequenced using the long-read HiFi (high fidelity) sequencing technology and a draft contig assembly of 1.57 Gb that represents 80.9% of the estimated genome size was produced. The assembly was highly repeat-rich with a repetitive DNA content of 81%. The assembly was phased into a primary and alternate assembly in order to minimize redundant contigs. Scaffolds were constructed with the primary assembly contigs, which were used for the identification of AMP (antimicrobial peptide) genes. Characteristic AMP domains and 3D structures were used to detect and verify antimicrobial peptides. As a result, 23 genes (PcAMP1-23) representing defensin, snakin, thionin, lipid transfer protein and vicilin-like AMP classes were identified. Bioinformatic analyses for the characterization of peptide physicochemical properties indicated that parsley AMPs are extracellular peptides, therefore, plausibly exert their antimicrobial effects through the most commonly described AMP action mechanism of membrane attack. AMPs are attracting increasing attention since they display their fast antimicrobial effects in small doses on both plant and animal pathogens with a significantly reduced risk of resistance development. Therefore, identification and characterization of AMPs is important for their incorporation into plant disease management protocols as well as medicinal research for the treatment of multi-drug resistant infections.

Characterization of alternate encounter assemblies of SARS-CoV-2 main protease

The assembly of two monomeric constructs spanning segments 1-199 (MPro1-199) and 10-306 (MPro10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ∼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro1-199 and MPro10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201-214 onto a second helical domain of MPro10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ∼6 μM MPro1-199. Catalytic activity arises primarily from MPro1-199 and is dependent on the interface interactions involving the N-finger residues 1-9 of MPro1-199 and E290 of MPro10-306. Importantly, our results confirm that a single N-finger region with its associated inter-subunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

Complex Pathways Drive Pluripotent Fmoc-Leucine Self-Assemblies.

Nature uses complex self-assembly pathways to access distinct functional non-equilibrium self-assemblies. This remarkable ability to steer same set of biomolecules into different self-assembly states is done by avoiding thermodynamic pit. In synthetic systems, on demand control over 'Pathway Complexity' to access self-assemblies different from equilibrium structures remains challenging. Here we show versatile non-equilibrium assemblies of the same monomer via alternate assembly pathways. The assemblies nucleate using non-classical or classical nucleation routes into distinct metastable (transient hydrogels), kinetic (stable hydrogels) and thermodynamic structures [(poly)-crystals and 2D sheets]. Initial chemical and thermal inputs force the monomers to follow different assembly pathways and form soft-materials with distinct molecular arrangements than at equilibrium. In many cases, equilibrium structures act as thermodynamic sink which consume monomers from metastable structures giving transiently formed materials. This dynamics can be tuned chemically or thermally to slow down the dissolution of transient hydrogel, or skip the intermediate hydrogel altogether to reach final equilibrium assemblies. If required this metastable state can be kinetically trapped to give strong hydrogel stable over days. This method to control different self-assembly states can find potential use in similar biomimetic systems to access new materials for various applications.

Complex Pathways Drive Pluripotent Fmoc‐Leucine Self‐Assemblies

AbstractNature uses complex self‐assembly pathways to access distinct functional non‐equilibrium self‐assemblies. This remarkable ability to steer same set of biomolecules into different self‐assembly states is done by avoiding thermodynamic pit. In synthetic systems, on demand control over ‘Pathway Complexity’ to access self‐assemblies different from equilibrium structures remains challenging. Here we show versatile non‐equilibrium assemblies of the same monomer via alternate assembly pathways. The assemblies nucleate using non‐classical or classical nucleation routes into distinct metastable (transient hydrogels), kinetic (stable hydrogels) and thermodynamic structures [(poly)‐crystals and 2D sheets]. Initial chemical and thermal inputs force the monomers to follow different assembly pathways and form soft‐materials with distinct molecular arrangements than at equilibrium. In many cases, equilibrium structures act as thermodynamic sink which consume monomers from metastable structures giving transiently formed materials. This dynamics can be tuned chemically or thermally to slow down the dissolution of transient hydrogel, or skip the intermediate hydrogel altogether to reach final equilibrium assemblies. If required this metastable state can be kinetically trapped to give strong hydrogel stable over days. This method to control different self‐assembly states can find potential use in similar biomimetic systems to access new materials for various applications.

Analysis of emergency assembly points for post-earthquake disaster management: a case study of Erzincan, Türkiye

The selection of emergency assembly points within the urban area holds significant importance for ensuring swift and effective intervention in the aftermath of a potential disaster until temporary shelter areas are prepared. In both historical and instrumental periods, Erzincan (Türkiye) has experienced major earthquakes resulting in significant loss of life and property. The likelihood of similar earthquakes occurring in Erzincan in the future remains high. Therefore, it is necessary to identify safe areas where people can assemble after an earthquake, moving away from hazardous zones. The aim of this study is to analyse the capacities and adequacies of emergency assembly points, which constitute a step in disaster management and enhance the earthquake resilience of the city, using Geographic Information Systems (GIS) based on selected criteria (accessibility, spatial distribution, proximity to healthcare facilities, size, capacity adequacy, and proximity to fault avoidance zones), and to propose solutions. The assessment revealed that the available assembly points are not homogeneously distributed throughout the city and are inadequate to meet the available building and population density. Therefore, in addition to the available 38 emergency assembly points, 67 new alternative emergency assembly points have been identified. The proposed emergency assembly points (in the 75–100% range) increased the number of neighborhoods within the 250 m service area by 43.75% and 42.5% for 500 m.The findings underscore the necessity for more comprehensive and effective planning for Erzincan in the event of a potential disaster or emergency.

Solution-Processed One-Dimensional Photonic Crystals Based on Hollow Silica Exhibiting High Refractive Index Contrast.

Poor interfacial quality and low refractive index contrast (Δn) are critical challenges for the development of high-performance one-dimensional photonic crystals (1DPhCs) via solution methods that impede their optical efficiency. Herein, we introduce an innovative approach by hybridizing hollow SiO2 with poly(vinyl alcohol), referred to as PHS, followed by alternate assembly with TiO2 via spin-coating, achieving a 1DPhC with Δn = 0.76 at the wavelength of 550 nm. This method circumvents the need for high-temperature treatment and complex curing conditions, resulting in a 1DPhC with superior interfacial and optical characteristics. By adjusting the thickness of the PHS layers, we can finely tune the reflectance spectrum, attaining over 99% reflectance at the photonic band gap. Furthermore, 1DPhC demonstrates excellent adhesion to polycarbonate substrates and retains its optimal optical performance even after rigorous environmental testing, including hygrothermal cycles, exposure to hot water, friction, and solvent sonication. This research paves the way for the facile fabrication of high-performance 1DPhCs under mild conditions, offering new perspectives for photonic material processing.

Alternate typhoid toxin assembly evolved independently in the two Salmonella species.

Typhoid toxin is an important Salmonella Typhi virulence factor and an attractive target for therapeutic interventions to combat typhoid fever. The recent discovery of a second version of this toxin has substantial implications for understanding S. Typhi pathogenesis and combating typhoid fever. In this study, we discover that a remarkably similar two-toxin paradigm evolved independently in Salmonella bongori, which strongly suggests that this is a critical aspect of typhoid toxin biology. We observe significant parallels between how the two toxins assemble and their capacity to intoxicate host cells during infection in S. Typhi and S. bongori, which provides clues to the biological significance of this unusual toxin arrangement. More broadly, AB5 toxins with diverse activities and mechanisms are essential virulence factors for numerous important bacterial pathogens. This study illustrates the capacity for novel A-B interactions to evolve and thus provides insight into how such a diverse arsenal of toxins might have emerged.

HSP90β controls NLRP3 autoactivation.

Gain-of-function mutations in NLRP3 are linked to cryopyrin-associated periodic syndromes (CAPS). Although NLRP3 autoinflammasome assembly triggers inflammatory cytokine release, its activation mechanisms are not fully understood. Our study used a functional genetic approach to identify regulators of NLRP3 inflammasome formation. We identified the HSP90β-SGT1 chaperone complex as crucial for autoinflammasome activation in CAPS. A deficiency in HSP90β, but not in HSP90α, impaired the formation of ASC specks without affecting the priming and expression of inflammasome components. Conversely, activating NLRP3 with stimuli such as nigericin or alum bypassed the need for SGT1 and HSP90β, suggesting the existence of alternative inflammasome assembly pathways. The role of HSP90β was further demonstrated in PBMCs derived from CAPS patients. In these samples, the pathological constitutive secretion of IL-1β could be suppressed using a pharmacological inhibitor of HSP90β. This finding underscores the potential of SGT1-HSP90β modulation as a therapeutic strategy in CAPS while preserving NLRP3's physiological functions.

DNA Polymerase-Steered Self-Propelled and Self-Enhanced DNA Walker for Rapid and Distinctly Amplified Electrochemical Sensing.

The development of a simple, rapid, easy-to-operate, and ultrasensitive DNA walker-based sensing system is challenging but would be very intriguing for the enormous applications in biological analysis and disease monitoring. Herein, a new self-propelled and self-enhanced DNA walking strategy was developed on the basis of a simple DNA polymerase-steered conversion from a typical alternate DNA assembly process. The sensing platform was fabricated easily by immobilizing only one hairpin probe (H1) and the sensing process was based on a simple one-step mixing with another hairpin-like DNA probe (H2) and DNA polymerase. The DNA polymerization could achieve target recycling and successive DNA walking steps. Interestingly, along with each DNA walking step, the new DNA walker sequence could be autonomously accumulated for a self-enhanced DNA walking effect. This provided a multilevel signal amplification ability for the ultrasensitive detection of the target with a low detection limit of 0.18 fM. Moreover, it could greatly reduce the reaction time with the sensing process finished within 1 h. The detection selectivity and the applicative potential in a complicated biological matrix were also demonstrated. Furthermore, the flexible control of sensing modes (self-enhanced DNA walking or the alternate DNA assembly) by using DNA polymerase or not offered a powerful means for sensing performance modulation. It thus opens a new avenue toward the development of a DNA walker-based sensing platform with both rapid and ultrasensitive features and might hold a huge potential for point-of-care diagnostic applications.

A hybrid metaheuristic based on the firefly and bat algorithms to solve the alternative subgraphs assembly line balancing problem

A hybrid metaheuristic based on the firefly and bat algorithms to solve the alternative subgraphs assembly line balancing problem

RELATIONSHIPS BETWEEN EMBODIED, OPERATIONAL, AND LIFE CYCLE CARBON IN PASSIVE HOUSE MULTIFAMILY RESIDENTIAL BUILDINGS

ABSTRACT The building sector is responsible for a significant portion of global CO2 emissions and any attempt to meet global climate change mitigation goals requires dramatic reductions in CO2 emissions from building construction and use. Among the many green building certification programs intended to reduce the environmental impact of buildings, the passive house standards are one of the most stringent certifications with respect to reducing operational energy. While there is significant research demonstrating the reductions in operational energy use in passive house and passive house inspired buildings, there is comparatively little research into the embodied energy and embodied carbon emissions associated with these buildings. The aim of this research was to evaluate the relationship between embodied carbon emissions, operational carbon emissions, and overall carbon use intensity in passive house certified residential buildings, using a recently completed, multifamily passive house as a case study. First, the case study building was defined, and a partial life cycle assessment (LCA) was performed to evaluate the embodied and operational carbon emissions associated with this base case. Second, a catalog of alternative wall and roof assemblies were defined and the LCA of the case study was updated for each alternative assembly to assess the impact on the embodied carbon, operational carbon, and overall carbon use intensity. The results of this analysis indicate that the material composition of the exterior envelope assemblies can have significant impact on the overall, cumulative carbon impact of a particular building. Moreover, the results also indicate that there are clear scenarios in which operational energy efficiency should be sacrificed in favor of reducing upfront embodied carbon emissions, particularly when evaluated over critical time spans. Based on these results, this study recommends that green building standards and certifications, including passive house but also USGBC LEED, Living Building Challenge, and others, should place greater emphasis on embodied carbon and holistic carbon accounting in addition to operational efficiency.

A new MIP approach for balancing and scheduling of mixed model assembly lines with alternative precedence relations

ABSTRACT In this paper, a new mixed integer programming (MIP) formulation is developed for balancing and scheduling of mixed model assembly lines with disjunctive precedence constraints among assembly tasks. To represent alternative precedence relations, AND/OR assembly graph was adopted. In case of alternative precedence relations, for each product multiple assembly plans exist, which can be represented by a set of alternative precedence subgraphs and only one of such subgraphs should be selected for each product. As the number of subgraphs exponentially increases with the number of disjunctive relations among the tasks, the computational complexity of simultaneous balancing and scheduling along with the assembly subgraph selection increases with the number of alternative precedence relations. Unlike the other MIP approaches known from the literature, the new model does not need the alternative assembly subgraphs to be to explicitly enumerated as input data and then used for indexing the variables. Instead, a new disjunctive precedence selection and task assignment variable and new constraints are introduced to optimally choose one relation for each subset of alternative precedence relations. The optimal solutions for computational examples of balancing and scheduling problems illustrate a superior performance of the new modelling approach.

Hybridizing Ti3C2Tx Layers with Layered Double Hydroxide Nanosheets at the Molecular Level: A Smart Electrode Material for H2O2 Monitoring in Cancer Cells.

Vertically stacked artificial 2D superlattice hybrids fabricated through molecular-level hybridization in a controlled fashion play a vital role in scientific and technological fields, but developing an alternate assembly of 2D atomic layers with strong electrostatic interactions could be much more challenging. In this study, we have constructed an alternately stacked self-assembled superlattice composite through integration of CuMgAl layered double hydroxide (LDH) nanosheets having positive charge with negatively charged Ti3C2Tx layers using well-controlled liquid-phase co-feeding protocol and electrostatic attraction and investigated its electrochemical performance in sensing early cancer biomarkers, i.e., hydrogen peroxide (H2O2). The molecular-level CuMgAl LDH/Ti3C2Tx superlattice self-assembly possesses superb conductivity and electrocatalytic properties, which are significant for obtaining a high electrochemical sensing aptitude. Electron penetration in Ti3C2Tx layers and rapid ion diffusion along 2D galleries have shortened the diffusion path and enhanced the charge transferring efficacy. The electrode modified with the CuMgAl LDH/Ti3C2Tx superlattice has demonstrated admirable electrocatalytic abilities in H2O2 detection with a wide linear concentration range and low real-time limit of detection (LOD) of 0.1 nM with signal/noise ratio (S/N) = 3. Practically, an electrochemical sensing podium based on the CuMgAl LDH/Ti3C2Tx superlattice has been effectively applied in real-time in vitro tracking of H2O2 effluxes excreted from different live cancer cells and normal cells after being encouraged by stimulation. The results exhibit that molecular-level heteroassembly holds great potential in electrochemical sensors to detect promising biomarkers.

The phased chromosome-scale genome of yellowhorn sheds light on the mechanism of petal color change

Yellowhorn (Xanthoceras sorbifolium), especially its varieties, is the red petal yellowhorn (X. sorbifolium var. purpurea), an important tree species with great ornamental value, and its flower petals change color throughout the flowering period. In this study, we mainly focused on the mechanism of the petal color change with transcriptomics and metabolomics data. A phased chromosome-scale assembly of the red petal yellowhorn genome was generated using the PacBio high-fidelity reads, Illumina short reads, and Phase genomics Proximo Hi-C data. The final de novo assembly yielded 533.67 Mb with a contig N50 of 5.42 Mb, and 27 501 protein-coding genes were predicted. Notably, an alternate haplotig assembly was also obtained. Furthermore, a variation database for the alleles within the genome was constructed. Subsequently, the expression pattern of flower pigmentation-related genes and allelic expression imbalance events were investigated. Apart from 6 genes involved in the anthocyanin biosynthesis pathway regulated by the activation of 15 MYB-bHLH-WD40s, the increased expression of senescence-related gene 1 (SRG1) and 2-oxoglutarate-dependent dioxygenase (DIOX5) might also result in decreasing content of lutein and increasing abundance of (E/Z)-phytoene, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside. These changes in genes and metabolites were most likely related to the petal color change in red petal yellowhorn. This phased chromosome-scale genome assembly provides more accurate genomic information for future molecular breeding and facilitates allele function studies of the red petal yellowhorn.

Chromosome-level, nanopore-only genome and allele-specific DNA methylation of Pallas's cat, Otocolobus manul.

Pallas's cat, or the manul cat (Otocolobus manul), is a small felid native to the grasslands and steppes of central Asia. Population strongholds in Mongolia and China face growing challenges from climate change, habitat fragmentation, poaching, and other sources. These threats, combined with O. manul's zoo collection popularity and value in evolutionary biology, necessitate improvement of species genomic resources. We used standalone nanopore sequencing to assemble a 2.5 Gb, 61-contig nuclear assembly and 17097 bp mitogenome for O. manul. The primary nuclear assembly had 56× sequencing coverage, a contig N50 of 118 Mb, and a 94.7% BUSCO completeness score for Carnivora-specific genes. High genome collinearity within Felidae permitted alignment-based scaffolding onto the fishing cat (Prionailurus viverrinus) reference genome. Manul contigs spanned all 19 felid chromosomes with an inferred total gap length of less than 400 kilobases. Modified basecalling and variant phasing produced an alternate pseudohaplotype assembly and allele-specific DNA methylation calls; 61 differentially methylated regions were identified between haplotypes. Nearest features included classical imprinted genes, non-coding RNAs, and putative novel imprinted loci. The assembled mitogenome successfully resolved existing discordance between Felinae nuclear and mtDNA phylogenies. All assembly drafts were generated from 158 Gb of sequence using seven minION flow cells.

A new design for the RF electron gun of the Iranian Light Source Facility (ILSF)

A new π mode RF electron gun is proposed for the Iranian Light Source Facility. This RF gun will be fabricated using a newly developed method based on clamping the parts together instead of vacuum brazing. In this method, RF contact and vacuum seal are mutually provided by a special copper gasket. The first section of this article describes the new π mode RF gun and its expected performance in detail. The report of our recent R&D tests on the new fabrication method is discussed in the next section. We performed the RF and vacuum measurements on a single-cell structure, and the copper gasket provided suitable RF contact, and a vacuum pressure of 2.5×10−7 mbar was also reached after 1 h of pumping with a turbo-molecular pump without baking. In the last section, we investigated using a high-current and low-emittance DC triode electron gun to be mounted on the RF gun as an alternative cathode assembly. The simulation results of this proposed scheme were compatible with our previous studies. The research and development of the new π mode RF electron gun and the proposed cathode assembly will begin soon.

Fast formation and assembly for spline‐based 3D fictitious domain methods

AbstractStandard finite element methods employ an element‐wise assembly strategy. The element's contribution to the system matrix is formed by a loop over quadrature points. This concept is also used in fictitious domain methods, which perform simulations on a simple tensor‐product background mesh cut by a boundary representation that defines the domain of interest.Considering such d‐dimensional background meshes based on splines of degree p with maximal smoothness, Cp−1, the cost of setting up the system matrix is 𝒪(p3d) per degree of freedom. Alternative assembly and formation techniques can significantly reduce this cost. In particular, the combination of (1) sum factorization, (2) weighted quadrature, and (3) row‐based assembly yields a cost of 𝒪(pd+1) for non‐cut background meshes. However, applying this fast approach to cut background meshes is an open challenge since they do not have a tensor‐product structure.This work presents techniques that allow the treatment of cut background meshes and thus the application of fast formation and assembly to fictitious domain methods. First, a discontinuous version of weighted quadrature is presented, which introduces a discontinuity into a cut test function's support. The cut region can be treated separately from the non‐cut counterpart; the latter can be assembled by the fast concepts. A three‐dimensional example investigates the accuracy and efficiency of the proposed concept and demonstrates its speed‐up compared to conventional formation and assembly.

RanBP9 controls the oligomeric state of CTLH complex assemblies

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8β subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8β and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8β-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures.