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bioRxiv2023

Sixteen oogenesin genes are dispensable for fertilityWhat is the significance of the dispensability of genes expressed in germ-cell?

Gene knockout experiments have shown that many genes are dispensable for a given biological function. The Oogenesin/Pramel family contains almost 85 paralogs, about thirty of which are specific to female (as well as male for some of them) germ cells. In this paper, we show that the deletion of a block of around 1Mb containing sixteen paralogous genes of the Oogenesin/Pramel family specific to germ cells, including Oogenesin-2, -3 and -4, has no consequences on fertility or prolificacy in mouse both sexes. The dispensability of these genes is probably due to the compensation by the other germ-cell specific paralogs.

molecular biology↗

bioRxiv2023

Analysis of DNA methylation in rainbow trout spermatozoa: the strengths and limitations of RRBS

DNA methylation is an important epigenetic mark in fish spermatozoa since it has been shown that some sperm methylome features are transmitted to the offspring. To ensure the transmission of unaltered information to the offspring, the characterization of this mark and its stability in spermatozoa is essential. DNA methylation status can be assessed at the whole genome level with an identification of the methylated and unmethylated cytosines using RRBS (reduced representation bisulfite sequencing). This method allows the sequencing of a subset of the genome expected to be enriched in CpGs. We aim to characterize the data provided by RRBS in rainbow trout spermatozoa, in order to evaluate the suitability of this approach for sperm biotechnologies studies. We observed that RRBS did provide a reduced amount of genomic data, thus allowing the processing of many biological replicates. Although, in our dataset, only a small fraction of the whole genome CpGs was present in all 6 to 12 replicates, the sum of the analyzed CpGs spanned 9 % of the total genomic CpGs. They distributed evenly all over the genome, and all genomic features were represented. RRBS is therefore an effective method to scan the DNA methylation of the along genome in a reduced pattern. However, one should be aware of the choices that are to be made regarding fragment size selection and regarding the options during bioinformatic data processing.

molecular biology↗

bioRxiv2023

Epigenetic and physiological alterations in zebrafish subjected to hypergravity

Gravity is one of the most constant environmental factors across Earths evolution and all organisms are adapted to it. Consequently, spatial exploration has captured the interest in studying the biological changes that physiological alterations are caused by gravity. In the last two decades, epigenetics has explained how the environmental cues are able to altered gene functions in the organisms. Although many studies addressed gravity, the underlying biological and molecular mechanisms that are occurred in altered gravity for those epigenetics-related mechanisms, are mostly inexistent. The present study addressed the effects of hypergravity on development, behavior, gene expression, and most importantly, on the epigenetic changes in a world-wide animal model, the zebrafish (Danio rerio). To perform hypergravity experiments, a custom-centrifuge simulating the large diameter centrifuge (100 rpm [~] 3 g) were designed and zebrafish embryos were exposed during 5 days post fertilization (dpf). Results showed a significant decrease of survival at 2 dpf but not significance in the hatching rate. Physiological and morphological alterations including fish position, movement frequency and swimming behavior showed significant changes due to hypergravity. Epigenetic studies showed a significant hypermethylation of the genome of the zebrafish larvae subjected to 5 days of hypergravity. A downregulation of the gene expression of three epigenetic-related genes (dnmt1, dnmt3, and tet1), although not significant, were further observed. Taken altogether, gravity alterations affected biological responses including epigenetics in fish, providing a valuable roadmap of the putative hazards of living beyond Earth.

cell biology↗

bioRxiv2023

The sensor of the bacterial histidine kinase CpxA is a novel dimer of extracytoplasmic Per-ARNT-Sim (PAS) domains

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA* alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologues revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation. SignificanceBacterial two-component systems play an essential role in sensing environmental cues, mitigating stress, and regulating virulence. We approach the study of a key Gram-negative sensor kinase CpxA with both classical methods in structural biology and genetic analysis and emerging protein-folding prediction software. This approach provides a wholistic perspective on the structure and function of histidine kinases as proteins with modular and cellular compartment-spanning domain architectures. We report a novel organization of PAS domains in CpxA, highlighting the versatility and diversity of this sensory fold. Ultimately, these studies will facilitate the continued development of novel antimicrobials against sensor kinases, including CpxA, which is a previously studied target for antimicrobials.

molecular biology↗

bioRxiv2018

Combinatorial recognition of clustered RNA elementsby a multidomain RNA-binding protein, IMP3

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

molecular biology↗

bioRxiv2019

Repertoire of morphable proteins in an organism

All living organisms have evolved to contain a set of proteins with variable physical and chemical properties. Efforts in the field of structural biology have contributed to uncovering the shape and the variability of each component. A set of experimental coordinates for a given protein can be used to define the \"morphness/unmorphness\". Here we show the results of global analysis of more than a thousand E. coli proteins, demonstrating that it would be a comprehensive method of understanding the evolved repertoire in an organism. By collecting \"UnMorphness Factor\" (UMF) determined for each of the proteins, the lowest and the highest boundaries of the experimentally observable structural variation are understood. The distribution of UMFs obtained for an organism is expected to represent how rigid and flexible components are balanced. The present analysis extends to evaluate the growing data from single particle cryo-electron microscopy, providing valuable information on effective interpretation to structural changes of proteins and the supramolecular complexes. The data and the method presented here also conform to FAIR data principles, having potential significance to advance the field of structural and molecular cell biology.

biophysics↗

bioRxiv2026

Ribosome stalling facilitates chloroplast targeting of nuclear-encoded proteins

Ribosomes translate mRNAs with variable elongation rates and frequently undergo transient stalling. Although ribosome stalling is known to regulate protein quality control and translational dynamics, its transcriptome-wide landscape and biological significance in plants remain largely unexplored. Here, using disome profiling in Arabidopsis thaliana, we generated a high-resolution map of ribosome stalling sites across the transcriptome and uncovered a marked enrichment on mRNAs encoding chloroplast-targeted proteins, particularly those involved in photosynthesis. We further found that ribosome stalling preferentially occurs when transit peptides emerge from the ribosome exit tunnel. Functional assays demonstrated that deletion of the stalling region significantly reduces chloroplast targeting efficiency. Together, our findings identify ribosome stalling as a regulatory layer that promotes efficient targeting of nuclear-encoded proteins to chloroplasts.

molecular biology↗

bioRxiv2023

Branched multimeric peptides as affinity reagents for detection of α-Klotho protein

-Klotho is a protein associated with aging that is expressed in the kidney, parathyroid gland, and choroid plexus. As a transmembrane protein, it acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. -Klotho has an extracellular domain that can be cleaved, released and circulated in the blood stream as a soluble form. Decreased levels of -Klotho are an indication of chronic kidney disease and other age-associated diseases. Detecting or labeling transmembrane and soluble -Klotho is a longstanding challenge that has impeded the in-depth understanding of its role. Here we describe branched multimeric peptides that recognize -Klotho with high affinity and selectivity in the biological milieu. The branched peptides are prepared in a single-shot synthesis by parallel automated fast-flow synthesis in under one hour. The branched -Klotho-binding peptides show improvement in affinity relative to the monomeric versions and can be used to label Klotho for live imaging in kidney cells. Our results demonstrate the potential of automated flow technology to deliver peptide-based reagents with complex architecture and improved affinity for the selective binding of target proteins in physiological settings.

molecular biology↗

bioRxiv2024

Genetic code expansion reveals site-specific lactylation in living cells reshapes protein function

Still in its infancy, the functions of lactylation remain elusive. To address this, we established a comprehensive workflow for lactylation studies that integrates the discovery of lactylation sites with proteomics, the expression of site-specifically lactylated proteins in living cells via genetic code expansion (GCE), and the evaluation of the resulting biological consequences. Specifically, we developed a wet-and-dry-lab combined proteomics strategy, and identified highly conserved lactylation at ALDOA-K147. Driven by its potential biological significance, we site-specifically expressed this lactylated ALDOA in mammalian cells and interrogated the biological changes. We discovered that it not only inhibited enzyme activity but also elicited gain-of-function effects----it dramatically reshaped the functionality of ALDOA by improving stability, enhancing nuclear translocation and affecting gene expression. Further, we demonstrated broad applicability of this workflow to study distinct histone lactylation sites. Together, we anticipate its wide uses in elucidating causative links between site-specific lactylation and target-centric or cell-wide changes.

molecular biology↗

bioRxiv2018

Transcription activity contributes to the activation of non-constitutive origins to maintain the robustness of S phase duration in African trypanosomes

The cosynthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences have not yet been investigated. Here, using equations and computational analysis we demonstrated that the number of constitutive origins mapped in the Trypanosoma brucei genome is close to the minimum required to complete replication within S phase duration. However, taking into account the location of these origins in the genome, the replication in due time becomes virtually impossible, making it necessary to activate non-constitutive origins. Moreover, computational and biological assays pointed to transcription being responsible for activating non-constitutive origins. Together, our results suggest that transcription action through conflicts with replication contributes to the firing of non-constitutive origins, maintaining the robustness of S phase duration. The usage of this entire pool of origins seems to be of paramount importance for the survival of this parasite that infects million people around the world since it contributes to the maintenance of the replication of its DNA.

molecular biology↗

bioRxiv2026

NaP-TRAP: A versatile and accessible workflow to dissect principles of translational regulation and mRNA stability

The translation of mRNA into protein is tightly regulated by both cellular trans-factors and cis-regulatory elements encoded within transcripts. Although transcript fate can be measured by transcript abundance or translation efficiency, separating the contribution of each individual cis-element within a single transcript is an ongoing challenge. Current massively parallel reporter assay (MPRAs) approaches enable systematic interrogation of cis-regulatory elements that control transcript stability, but translation-focused MPRAs remain technically limited and often inaccessible. Here we present Nascent Peptide Translating Ribosome Affinity Purification (NaP-TRAP), a reporter-based approach that simultaneously measures translation and mRNA abundance. Unlike previous methods, NaP-TRAP captures translation directly through the immunoprecipitation of epitope-tagged nascent peptide chains, providing instantaneous, frame-specific readouts without specialized instrumentation. The method is highly scalable from single reporters to complex libraries, and adaptable across in vivo and in vitro systems. NaP-TRAP is versatile, allowing assessment of cis-regulatory impact of elements distributed throughout the mRNA, from cap-to-tail. This protocol covers experimental design, reporter construction, sample processing, and computational analysis for both low- and high-throughput applications. Bench work can be completed in 4- 5 days, with qPCR-based readouts requiring only basic Excel skills for data processing. Sequencing-based readouts require skills in command-line tools and Python scripting and add an additional 2-3 days. NaP-TRAP thus offers an accessible, robust, and quantitative platform to decode the regulatory logic of mRNA translation and stability in diverse biological contexts. Basic Protocol 1Design, assembly, and synthesis of NaP-TRAP reporter libraries. Support Protocol 1Design, assembly, and synthesis of NaP-TRAP individual reporters and spike-ins. Basic Protocol 2NaP-TRAP delivery by micro-injection in zebrafish embryos. Alternate Protocol 1NaP-TRAP delivery by transfection in cultured mammalian cells. Basic Protocol 3NaP-TRAP pulldown and RNA extraction. Basic Protocol 4Preparation of NaP-TRAP cDNA Sequencing Libraries. Alternate Protocol 2NaP-TRAP-qPCR module for low-cost validation. Basic Protocol 5Computational analysis of NaP-TRAP MPRA data.

molecular biology↗

bioRxiv2026

Protein Function Prediction with Pretrained ProtT5 Embeddings and Gradient Boosting

Protein function prediction remains a central challenge in computational biology due to the extreme sparsity and long-tail distribution of Gene Ontology (GO) [1] annotations. Advances in protein language models enable the extraction of dense, fixed-length representations from amino acid sequences, offering a scalable alternative to hand-picked features such as physicochemical properties. In this work, we evaluate a transformer-based embedding approach using ProtT5-XL combined with classical and modern multi-label classifiers for Gene Ontology prediction in the CAFA-6 setting. Fixed-length embeddings were generated via mean pooling of transformer hidden states and used as input to one-vs-rest logistic regression, gradient-boosted decision trees, and a neural network. Models were evaluated on held-out validation data with a focus on threshold selection, prediction sparsity, and behavior across frequent and rare GO terms. Gradient boosting consistently provided the best balance between predictive performance and stable prediction behavior, motivating its use for ontology-specific predictors across molecular function, biological process, and cellular component annotations. This study highlights practical modeling choices for large-scale protein function prediction using pretrained sequence embeddings and provides an interpretable baseline for future CAFA evaluations.

bioinformatics↗

bioRxiv2020

Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying essential circRNAs

Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for functional circRNAs. To address this issue, we leveraged the CRISPR/Cas13d system2 for circRNAs functional screenings. We optimized a strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNAs screening yielded a high rate of false positives phenotypes, while optimized CRISPR/Cas13d led to the identification of bona-fide functional circRNAs. Collectively, we developed a specific and reliable approach to functionalize circRNAs in a high-throughput manner.

molecular biology↗

bioRxiv2021

MRG proteins are shared by multiple protein complexes with distinct functions

MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain recognizing H3K36me3 in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing and DNA repair by homologous recombination. In order to get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the chromodomain, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase and PALB2/BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR-tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that can specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding chromodomain of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes which is essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes. HighlightsO_LIMRG15 and MRGX are stably associated with several different protein complexes important for genome expression and stability. C_LIO_LISeveral MRG-containing complexes are chromatin modifiers. C_LIO_LISpecific point mutations in the MRG domain differentially affect associated complexes. C_LIO_LIA major human complex homologous to the yeast TINTIN complex is identified. C_LIO_LIThe protein EP400NL competes with EP400 to functionally separate TINTIN from the NuA4/TIP60 complex. C_LIO_LITINTIN contains a bromodomain and a chromodomain to regulate transcription. C_LI

molecular biology↗

bioRxiv2023

Unraveling Coinfection Dynamics into 100 Whole Genome of Diarrheal Pathogens: A Genome-to-Systems Biology Approach with Plesiomonas shigelloides

Diarrhea is the second leading cause of mortality among infants under the age of five. One of the main causes of this disease is multipathogenic infections, which can make the conditions of patients even worse. Plesiomonas shigelloides (P. shigelloides) is one of the pathogenic bacteria that contributes to the pathophysiology of diarrhea and may be implicated in coinfection with other diarrheal pathogens. Therefore, the purpose of this study is to investigate the hypothetical proteins to explore the genetic insights of P. shigelloides and its relationships with common diarrheal diseases. For this reason, we used 16S rRNA sequencing together with several biochemical tests to identify the bacteria that we isolated from diarrheal patients (8 years). Afterwards, the whole genome of P. shigelloides was sequenced, assembled and annotated in order to obtain the genomic insights of P. shigelloides. In addition, the common virulence genes of ten (10) common diarrhea-causing bacteria were identified from 100 whole genome sequences. Finally, the system biology approach was applied to predict the coinfection pattern between P. shigelloides and the virulence genes of 10 bacteria. The complete genome sequencing analysis of this bacterium revealed 899 hypothetical proteins from which 33 hypothetical proteins shared the clusters with the 109 virulence genes of 10 distinct diarrheal pathogens, forming a strong interaction based on biological processes, molecular functions, subcellular localization, or pathways. All diarrhea causing bacteria were found to have P. shigelloides microbial interactions; however, V. cholerae exhibited the strongest relationships, while C. difficile showed the weakest. The results of this investigation clearly imply that P. shigelloides shares a coinfection pattern with other bacteria that cause diarrhea. Finally, the findings from the complete genome provide new avenues for researchers to pursue their investigation of the pathophysiology of diarrhea.

systems biology↗

bioRxiv2025

The break in the cycle: Inositol pyrophosphate fluxomics disentangled via mathematical modelling

This study investigates the metabolic pathways of inositol pyrophosphates (IPPs) in the yeast cell line {Delta}SPX and the human tumor cell line HCT116. Utilizing pulse-labelling experiments with 18O water and ordinary differential equation (ODE) models, we explore the synthesis and turnover of the highly phosphorylated IPP, 1,5-InsP8. Our findings challenge the notion that 1,5-InsP8 can be synthesized through distinct routes, revealing a linear reaction sequence in both systems. Employing model reduction via the profile likelihood method, we achieved statistically concise identifiability analysis that led to significant biological insights. In yeast, we determined that 1,5-InsP8 production primarily occurs through the phosphorylation of 5-InsP7, with the pathway involving 1-InsP7 deemed unnecessary as its removal did not compromise model accuracy. In HCT116 cells, 1,5-InsP8 synthesis is mainly driven by 1-InsP7, with variations observed across different experimental conditions. These results underscore the utility of model reduction in enhancing our understanding of metabolic pathways, challenging traditional views of IPP metabolism, and providing a framework for future investigations into the regulation and implications of linear IPP pathways in eukaryotic cells.

molecular biology↗

bioRxiv2022

Metabolic-imaging of human glioblastoma explants: a new precision-medicine model to predict tumor treatment response early

BackgroundGlioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) treatment, and radiotherapy (RT) remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical approaches that do not fully respect the complexity of GB cell biology and fail to test efficiently anti-cancer treatments. Therefore, better treatment screening approaches are needed. In this study, we have developed a novel functional precision medicine approach to test the response to anticancer treatments in organoids derived from the resected tumors of glioblastoma patients. MethodsGB organoids were grown for a short period of time to prevent any genetic and morphological evolution and divergence from the tumor of origin. We chose metabolic imaging by NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to predict early and non-invasively ex-vivo anti-cancer treatment responses of GB organoids. TMZ was used as the benchmark drug to validate the approach. Whole-transcriptome and whole-exome analyses were then performed to characterize tumor cases stratification. ResultsOur functional precision medicine approach was completed within one week after surgery and two groups of TMZ Responder and Non Responder tumors were identified. FLIM-based metabolic tumor stratification was well-reflected at the molecular level, confirming the validity of our approach, highlighting also new target genes associated with TMZ treatment and identifying a new 17 gene molecular signature associated with survival. The number of promoter methylated tumors for the MGMT gene was higher in the responsive group, as expected, however, some non-methylated tumor cases turned out to be nevertheless responsive to TMZ, suggesting that our procedure could be synergistic with the classical MGMT methylation biomarker. ConclusionsFor the first time, FLIM-based metabolic imaging was used on ex-vivo live glioblastoma organoids. Unlike other approaches, ex-vivo patient-tailored drug response is performed at an early stage of tumor culturing with no animal involvement and with minimal tampering with the original tumor cytoarchitecture. This functional precision medicine approach can be exploited in a range of clinical and laboratory settings to improve the clinical management of GB patients and implemented on other cancers as well.

molecular biology↗

bioRxiv2021

Diversification of Transcription Factor NF-κB in Protists

In this report, we investigate the evolution of transcription factor NF-{kappa}B by examining its structure, activity, and regulation in two protists using phylogenetic, cellular, and biochemical techniques. In Capsaspora owczarzaki (Co), we find that full-length NF-{kappa}B has an N-terminal DNA-binding domain and a C-terminal Ankyrin (ANK) repeat inhibitory domain, and its DNA-binding activity is more similar to metazoan NF-{kappa}B rather than Rel proteins. As with mammalian NF-{kappa}B proteins, removal of the ANK repeats is required for Co-NF-{kappa}B to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-{kappa}B is not induced by co-expression of IKK in human cells. Exogenously expressed Co-NF-{kappa}B localizes to the nucleus in Co cells. NF-{kappa}B mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-{kappa}B in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-{kappa}B. We also show that three NF-{kappa}B-like proteins from the choanoflagellate Acanthoeca spectabilis (As) all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-{kappa}B, and lack C-terminal ANK repeats. All three As-NF-{kappa}B proteins constitutively enter the nucleus of human and Co cells, but differ in their DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-{kappa}B proteins can form heterodimers, indicating that NF-{kappa}B diversified into multi-subunit families at least two times during evolution. Overall, these results present the first functional characterization of NF-{kappa}B in a taxonomic kingdom other than Animalia and provide information about the evolution and diversification of this biologically important transcription factor. SignificanceThese results represent the first functional characterization of the biologically important transcription factor NF-{kappa}B in a taxonomic kingdom other than Animalia. As such, they provide information on the evolutionary origins and basal diversification of NF-{kappa}B outside of metazoans. These results suggest that NF-{kappa}B plays life stage-specific roles in Capsaspora, the closest unicellular ancestor to all metazoans. Finally, the analysis of three NF-{kappa}B proteins in a single choanoflagellate indicates that choanoflagellates have subclasses of NF-{kappa}Bs, which can form heterodimers, suggesting that NF-{kappa}B subunit expansion and diversification has occurred at least twice in evolution.

cell biology↗