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Results for “molecular biology”

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bioRxiv2024

m6A methylation dynamically participates in the immune response against Vibrio anguillarum in half-smooth tongue sole (Cynoglossus semilaevis)

N6-methyladenosine (m6A) is the most prevalent RNA modification and a multifaceted regulator capable of affecting various aspects of mRNA metabolism, thereby playing important roles in numerous physiological processes. However, it is still unknown whether, when, and to what extent m6A modulation are implicated in the immune response of an economically important aquaculture fish, half-smooth tongue sole (Cynoglossus semilaevis). Herein, we systematically profiled and characterized the m6A epitranscriptome and transcriptome in C. semilaevis after the infection of Vibrio anguillarum, a serious threat to the aquaculture industry. We demonstrated that m6A could be modulated as early as 4-hour post infection (hpi), and the overall intensity of m6A methylation was enhanced following infection. Both conservative and novel motifs were uncovered from the m6A modification sites. Furthermore, differentially m6A methylated genes (DMGs) and differentially expressed genes (DEGs) were identified, and functional enrichment revealed multiple immune-related pathways, especially the FoxO signaling pathway which showed significance in every comparison. Joint analysis highlighted the remarkedly dynamic role of m6A on gene expression, i.e. early on, m6A mainly prioritized the down-regulation of specific genes, and later, it switched gears to promote expression of another set of genes. Moreover, key immune-related genes, including pdp1, rgs5l, and plk2b, were identified. To our limited knowledge, this is the first study comprehensively characterizing the global m6A atlas in aquaculture fish species. The presented results provide new insights into the dynamics of m6A modifications in the transcriptome of the half-smooth tongue sole following bacterial infection. Further studies are warranted to elucidate the functional significance of these changes and to understand how they affect specific biological processes.

molecular biology↗

bioRxiv2025

Functional and phylogenomic approaches reveal novel types of M42 peptidases with contrasted enzymatic properties in Archaea

M42 peptidases are half-megadalton aminopeptidases characterized by a tetrahedral architecture (TET) ubiquitous across all domains of life. Despite their widespread occurrence, their evolutionary history and functional diversity remain largely unexplored. Here we show an unsuspected and largely untapped wealth of archaeal TET peptidases, exhibiting remarkable functional heterogeneity, as illustrated by the characterization of six novel enzymes. Using structural biology, phylogeny, and enzymatic studies, we establish robust criteria for high-throughput identification of TET peptidases and perform the first systematic study of their genomic distribution and functional diversity across the archaeal kingdom. We propose an 11-group classification for these enzymes and identify one group as the ancestral lineage that first emerged in Archaea. By coupling taxonomic distribution patterns with functional insights, we highlight the presence of multiple TET enzymes with selective activities in heterotrophic and mixotrophic organisms, suggesting a role for TET peptidases in the degradation of environmental peptides. Overall, this work illuminates the underexplored diversity of TET enzymes, uncovering a complex evolutionary history linked to their potential biological function.

molecular biology↗

bioRxiv2025

Single-cell visual proteomics of a minimal bacterium reveals structural coordination in gene expression

Translation is a central process in gene expression. Regulation of this process is complex, depends on factors that include cell state and the subcellular environment, and is subject to modulation via crosstalk to processes like transcription or translocation. Here, we used cryo-electron tomography of native and antibiotic-perturbed Mycoplasma pneumoniae cells to resolve 140 maps that recapitulate bacterial translation during the initiation, elongation, termination, and recycling phases. We visualized multiple transcription-translation complexes, allowing us to propose a threading-based translation reinitiation mechanism, and to provide structural evidence for a long-hypothesized supercomplex that coordinates transcription, translation, and membrane attachment. We further resolved abundant membrane-associated large ribosomal subunits, and suggest that dissociation from membranes depends on the conditional initiation of new translation, consistent with a conserved mechanism in mammalian cells. This work visualizes the multilayered control of bacterial translation and demonstrates the power of in-cell structural biology to investigate regulatory circuits in gene expression.

molecular biology↗

bioRxiv2019

iDamage: a method to integrate modified DNA into the yeast genome

In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the yeast genome, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of rad51 recombinase) or through its control by preventing the poly-ubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of TLS. Such regulatory aspects of the DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will allow exploration of many biological processes related to replication and repair of modified DNA.

molecular biology↗

bioRxiv2026

Molecular Translators as a Computational Primitive for Biomarker Discovery: Learnability Gains Under Conserved Information Ceilings

Virtual molecular mapping systems such as MISO and GigaTIME introduce a potentially transformative primitive in computational pathology: translation of H&E whole-slide images into biologically structured molecular representations, learned on paired cohorts and deployed as an inference-time map. Despite sustained progress in machine learning, H&E-to-molecular-biomarker (e.g., gene mutation) prediction continues to exhibit recurrent field-level performance plateaus whose drivers remain poorly resolved. It remains unclear whether continued optimization targets a removable methodological limitation or instead presses against an intrinsic ceiling imposed by morphology. We develop a formal framework characterizing what deterministic translators can and cannot change. Histology-based biomarker modeling is governed by two constraints: method-limited gaps (finite labels, weak supervision, structured nuisance) and modality-limited ceilings (intrinsic slide-specific information in morphology). Because deterministic translation introduces no new slide-level measurements at inference, H&E information ceilings are conserved; however, translation can still improve finite-sample learnability, yielding an apparent information-performance paradox that we formalize as learnability gains under conserved information ceilings. We derive falsifiable signatures distinguishing these regimes and characterize them in controlled analytical experiments anchored to representative systems, including MISO and GigaTIME. We introduce an open-source toolkit comprising learning regime diagnosis, information-ceiling estimations, phase analyses, fidelity perturbation tests, and shortcut-confounding stress tests as an operational rubric for identifying and overcoming removable performance plateaus in translator-assisted molecular biomarker discovery and computational pathology.

bioinformatics↗

bioRxiv2020

Identifying spawning activity in aquatic species based on environmental DNA spikes

An understanding of the reproductive biology of aquatic organisms is crucial for the efficient conservation and management of species and/or populations. Nevertheless, conventional spawning surveys such as visual- and capture-based monitoring generally require laborious, time-consuming work and are subject to monitoring biases such as observer bias, as well as miscounts due to false spawning. In addition, direct capture often damages eggs or individuals. Thus, an efficient non-invasive approach for monitoring spawning activity on aquatic species would be a valuable tool to understand their reproductive biology and conserving biodiversity. Here, we proposed an environmental DNA (eDNA)-based approach for monitoring and understanding spawning activity by observing spikes in eDNA concentration after spawning activity. We found in cross experiments using two medaka species (Oryzias latipes and O. sakaizumii, 1:1 individual per tank) that an eDNA spike occurred in only male species after spawning activity. In addition, the magnitude of the eDNA spike was dependent on the number of spawning activities with egg and sperm release. In the field survey during the reproductive season, eDNA concentration after spawning were 3-25 times higher than before spawning. On the other hand, there was no increase in eDNA concentration during the non-reproductive season. Therefore, our results demonstrated that spikes in the eDNA concentration are mainly caused by the release of sperm during spawning activity, and it can be used as evidence of spawning in field survey. The presented approach could be a practical tool for studying reproductive biology and provides an opportunity to design effective conservation and environmental management actions.

molecular biology↗

bioRxiv2021

Secondary structure of subgenomic RNA M of SARS-CoV-2

SARS-CoV-2 belongs the Coronavirinae family. As other coronaviruses, SARS-CoV-2 is enveloped and possesses positive-sense, single-stranded RNA genome of [~] 30 kb. Genome RNA is used as the template for replication and transcription. During these processes, positive-sense genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) are created. Several studies showed importance of genomic RNA secondary structure in SARS-CoV-2 replication. However, the structure of sgRNAs have remained largely unsolved so far. In this study, we performed probing of sgRNA M of SARS-CoV-2 in vitro. This is the first experimentally informed secondary structure model of sgRNA M, which presents features likely to be important in sgRNA M function. The knowledge about sgRNA M provides insights to better understand virus biology and could be used for designing new therapeutics.

molecular biology↗

bioRxiv2022

Plant phase extraction (PPE): A novel method for enhanced discovery of RNA-binding proteome in plants

RNA-binding proteins (RBPs) are versatile effectors in posttranscriptional gene regulation. Systematic profiling of RBPs in plants has been limited to proteins interacting with polyadenylated (poly(A)) RNAs due to the lack of an efficient method of isolating RBPs associated with non-poly(A) RNAs. Here we reported the establishment and application of plant phase extraction (PPE) as a novel method to comprehensively discover the RNA-binding proteome in Arabidopsis leaf tissues, leading to the isolation of 1,169 RBPs, of which 673 corroborate with previously reported RBPs and 496 are novel RBPs. PPE showed unmatched ability in capturing 374 diverse RNA-binding domains (RBDs), while 44% of the RBPs lack recognized RBDs. PPE recovered far more ribosomal and tRNA-related RBPs and metabolic enzymes with moonlighting RBP functions than any other individual report from plants, signifying its advantage in unbiasedly retrieving non-poly(A) and unconventional RBPs. We proposed that intrinsically disordered regions (IDRs) contributed to the non-classical binding of some novel RBPs, and provided the first evidence that enzymatic domains from metabolic enzymes have additional roles in RNA-binding. Taken together, our findings demonstrated that PPE is an effective approach to identifying a wide range of RBPs in complex plant tissues and may have broad biological implications.

molecular biology↗

bioRxiv2023

Rapid and sensitive detection of native glycoRNAs

Chemical tools enable precise characterization of many biopolymers, including glycoconjugates. Metabolic chemical reporters enabled the discovery of glycoRNAs, however they have certain limitations due the requirement of having living cells to incorporate the modified sugar. Here we develop a periodate oxidation and aldehyde ligation method to detect and characterize native sialoglycoRNAs, termed rPAL. With optimized RNA biochemistry to enhance recovery and analysis of small RNAs, we show rPAL is at least an order of magnitude more sensitive than previous methods for detecting sialoglycoRNAs. These improvements allow rPAL to detect sialoglycoRNA from human clinical samples as demonstrated by defining the abundance and patterns of sialoglycoRNAs from sorted populations of peripheral blood mononuclear cells. The sensitivity, robustness, and flexibility of rPAL will allow greater access towards characterizing glycoRNA biology.

molecular biology↗

bioRxiv2023

Structure-function analysis of the cyclic beta-1,2-glucan synthase

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic {beta}-1,2-glucan (C{beta}G) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). C{beta}G is produced by the cyclic glucan synthase (Cgs), a massive multi-domain membrane protein. So far, its structure as well as the mechanisms underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We were able to determine the structure of this complex protein machinery and clarify key aspects of C{beta}G synthesis. Our research opens new possibilities for combating pathogens that rely on polysaccharide virulence factors and can lead to new synthetic biology approaches for producing complex cyclic sugars.

molecular biology↗

bioRxiv2024

RNA dicing regulates the expression of an oncogenic JAK1 isoform

mRNA transcripts have limited potential for protein synthesis, defined by their open reading frames1. However, recent advances have revealed a far more complex reality, in which the proteome exceeds the perceived limits of the transcriptome2-6 exposing a significant gap in our understanding. Our prior studies have demonstrated that mRNA can undergo further processing, yielding truncated, uncapped mRNAs with translation potential2,7. Yet, the biological importance of this process remains mostly unclear. Here, we demonstrate that cleavage within the coding sequence of JAK1 mRNA produces an uncapped variant downstream to the cleavage site, at the expense of the full-length transcript. This results in the independent translation of the JH1 kinase domain, a process we term RNA dicing. Notably, canonical and diced JAK1 variants have distinct impacts on cell proliferation and tumorigenesis, operating independently, localizing to distinct cellular compartments. In addition, the activation of JAK1 signaling through IFN{gamma} induction promotes the dicing of JAK1, thereby altering the balance of isoforms present. Base editor screens reveal that stop-codon nonsense mutations, which are typically considered loss-of-function, have differing impacts depending on their position relative to the dicing site. In agreement with this, JAK1 nonsense frameshift (JAK1fs) mutations in endometrial tumors inhibit the tumor-suppressive functions of canonical JAK1, while amplifying the oncogenic potential of the diced JH1 kinase domain. Lastly, we demonstrate that Momelotinib8, a JAK1-specific inhibitor, is more effective in cancer cells carrying a "loss-of-function" JAK1fs mutation, highlighting the significant impact of RNA dicing biology as a potent tool for patient stratification. Our findings characterize RNA dicing as a fundamental regulatory machinery that diversifies the potential products of a single mRNA molecule, and allows for significant variation in biological function.

molecular biology↗

bioRxiv2024

The structural basis for RNA slicing by human Argonaute2

Argonaute (AGO) proteins associate with guide RNAs to form complexes that slice transcripts that pair to the guide. This slicing drives post-transcriptional gene-silencing pathways that are essential for many eukaryotes and the basis for new clinical therapies. Despite this importance, structural information on eukaryotic AGOs in a fully paired, slicing-competent conformation--hypothesized to be intrinsically unstable--has been lacking. Here we present the cryogenic-electron microscopy structure of a human AGO-guide complex bound to a fully paired target, revealing structural rearrangements that enable this conformation. Critically, the N domain of AGO rotates to allow the RNA full access to the central channel and forms contacts that license rapid slicing. Moreover, a conserved loop in the PIWI domain secures the RNA near the active site to enhance slicing rate and specificity. These results explain how AGO accommodates targets possessing the pairing specificity typically observed in biological and clinical slicing substrates.

molecular biology↗

bioRxiv2025

Nuclear RNA exosome targeting onto chromatin loci in the degradation of unstable transcripts affects transcriptional programs in liver cells.

HP1, a hallmark of pericentromeric heterochromatin, is a chromatin-bound regulator of co-transcriptional processes including alternative splicing, but its role in RNA degradation remains unexplored. Here, we uncover a direct interaction between HP1 and the RNA exosome, a major RNA decay complex. In mouse embryonic liver cells, inactivation of all three HP1 isoforms led to accumulation of retrotransposon-derived RNAs and stabilization of enhancer RNAs. These changes coincided with increased activity at a subset of liver enhancers particularly sensitive to reduced exosome activity, many of which regulate genes encoding extracellular matrix components such as Col6a1 and Col6a2. Stratifying hepatocellular carcinoma samples by HP1 expression further revealed that tumors with low HP1 were marked by reduced RNA degradation, and increased expression of a similar subset of genes encoding extracellular matrix components and possibly contributing to tumor stiffness. These results suggest that HP1s impact on RNA turnover contributes to its function in cancer biology.

molecular biology↗

bioRxiv2025

Accessible and fast single-cell proteomics by DIA integrating Tecan UNO cell dispensing platform, Vanquish Neo LC and Exploris 480 MS

Recent technological advancements in high-sensitivity LC-MS platforms and MS acquisition strategies have addressed key analytical challenges in single-cell proteomics (SCP), paving the way for SCP to become a transformative tool in biology and medicine. Techniques such as MS2 DIA and HRMS1 DIA exploit wide isolation windows and high-resolution MS1 and MS2 spectra to maximize protein identifications, even from low-abundance signals. In parallel, improved data processing algorithms reduce missing values and enhance the reliability of SCP data. These advancements are complemented by automated cell isolation technologies, which ensure precise single-cell handling with minimal sample loss, and in some implementations enable integrated, automated downstream sample preparation steps. Despite this progress, practical, end-to-end best practices for MS-based SCP remain an active area of optimization. Severe sample limitation, adsorption-related losses, the need for robust quantification across hundreds to thousands of single cells, and the lack of comprehensive protocols that systematically integrate and compare critical workflow variables complicate method selection and SCP implementation. In this context, this study builds on recent LC-MS based SCP advancements by presenting a complete, optimized, minimum user operation SCP workflow that integrates the Tecan UNO cell dispensing platform, a Vanquish Neo LC system, and an Orbitrap Exploris 480 mass spectrometer, applied to human primary cells. The workflow is designed to minimize data/cell loss, ensure high reproducibility, and maximize identifications. We evaluate key parameters, including acquisition schemes and LC gradient and column, and distill practical recommendations. The resulting protocol provides a foundation for an easy and accessible routine SCP application in biology and medicine.

molecular biology↗

bioRxiv2018

Interaction pathways promote spliceosome module integration and network-level robustness to cascading effects

The functionality of distinct types of protein networks depends on the patterns of protein-protein interactions. A problem to solve is understanding the fragility of protein networks to predict system malfunctioning due to mutations and other errors. Spectral graph theory provides tools to understand the structural and dynamical properties of a system based on the mathematical properties of matrices associated with the networks. We combined two of such tools to explore the fragility to cascading effects of the network describing protein interactions within a key macromolecular complex, the spliceosome. Using S. cerevisiae as a model system we show that the spliceosome network has more indirect paths connecting proteins than random networks. Such multiplicity of paths may promote routes to cascading effects to propagate across the network. However, the modular network structure concentrates paths within modules, thus constraining the propagation of such cascading effects, as indicated by analytical results from the spectral graph theory and by numerical simulations of a minimal mathematical model parameterized with the spliceosome network. We hypothesize that the concentration of paths within modules favors robustness of the spliceosome against failure, but may lead to a higher vulnerability of functional subunits which may affect the temporal assembly of the spliceosome. Our results illustrate the utility of spectral graph theory for identifying fragile spots in biological systems and predicting their implications.

molecular biology↗

bioRxiv2018

Proteomic analysis of RNA-dependent chromatin association of nuclear proteins

Various coding and non-coding transcripts are known to associate with chromatin and now there is accumulating evidence that interaction between RNA-binding proteins (RBPs) and RNA molecules regulate not only co-transcriptional mRNA processing, but also other biological processes within the nucleus. Although over a thousand of RBPs have been identified by several mass spectrometry-based methods, it is still unclear which of these RBPs actually associate with chromatin, especially through interaction with RNAs. In addition, biological outcomes of such RBP-RNA-chromatin interactions are yet to be elucidated.\n\nHere we describe a simple proteomics-based method for systematic screening of RBPs that are anchored to chromatin and/or insoluble nuclear substructures by RNA molecules. We used RNase A to release such RBPs from chromatin fraction and analyzed RNase A-solubilized proteins by mass spectrometry. Using this method, we were able to identify 156 RNase A-solubilized proteins of which 144 were known RBPs/RBP candidates. Interestingly, several key players of the non-homologous end-joining (NHEJ) pathway were enriched in RNase A-solubilized fraction and the RNA-mediated chromatin association of these factors appeared to be dependent on transcriptional elongation. Furthermore, some enzymes involved in metabolic pathways were also released from chromatin and/or an insoluble nuclear structure by RNase A treatment. In summary, our methodology is highly versatile and is potentially a useful tool to unravel new biological functions for RBP-RNA-chromatin interactions.

molecular biology↗

bioRxiv2019

Origin of cooperativity in the activation of dimeric transcription factors

Cooperative behavior in the binding of ligands to a protein is often viewed as a complex phenomenon where conformational changes induced by the binding of the first ligand leads to tighter binding of subsequent ligands. We revisit the ligand-dependent activation of dimeric transcription factors and show that this process may appear cooperative even when it results from independent lig- and binding events. This effect is further accentuated through binding of the activated transcription factor to its cognate operator site on the DNA, where we demonstrate that cooperative activation is a stable fixed point. Our analysis nicely accounts for the apparent co-operativity inherent in the biological activity of many dimeric transcription factors.

molecular biology↗

bioRxiv2019

Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replicated three-molecule RNA progeny of Brome mosaic virus (BMV) are packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasi-symmetry. Type 1 (B1v) and type 2 (B2v) virions respectively package genomic RNA1 or RNA2, while type 3 (B3+4v) co-packages genomic RNA3 (B3) and its sub-genomic RNA4 (B4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Physical and biochemical approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types, so protease-based mapping experiments were used to analyze the conformational dynamics of the individual virions. The crystallographic structure of the BMV capsid shows four trypsin-cleavage sites (K65, R103, K111 and K165 on the A, B and C subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1v and B2v released only two peptides involving amino acids 2-8 and 16-22 from the N-proximal arginine-rich RNA binding motif. In contrast, B3+4v capsids are unstable to trypsin, releasing several peptides in addition to the four sites predicted to be exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids. IMPORTANCEThe majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that in solution virions are highly dynamic assemblies. The three genomic RNAs (RNAs 1, 2 and 3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the capsid dynamics by MALDI-TOF analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNAs1 and 2 are more stable and dynamically distinct from those co-packaging RNA3 and 4, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

molecular biology↗