SEARCH · bioRxiv Science

Results for “molecular biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,207 records · Page 67Linked to original sources

bioRxiv2023

Inverse-folding design of yeast telomerase RNA increases activity in vitro

Saccharomyces cerevisiae telomerase RNA, TLC1, is an 1157 nt non-coding RNA that functions as both a template for DNA synthesis and a flexible scaffold for telomerase RNP holoenzyme protein subunits. The tractable budding yeast system has provided landmark discoveries about telomere biology in vivo, but yeast telomerase research has been hampered by the fact that the large TLC1 RNA subunit does not support robust telomerase activity in vitro. In contrast, 155-500 nt miniaturized TLC1 alleles comprising the catalytic core domain and lacking the RNAs long arms do reconstitute robust activity. We hypothesized that full-length TLC1 is prone to misfolding in vitro. To create a full-length yeast telomerase RNA predicted to fold into its biological relevant structure, we took an inverse RNA folding approach, changing 59 nucleotides predicted to increase the energetic favorability of folding into the modeled native structure based on the p-num feature of Mfold software. The sequence changes lowered the predicted {triangleup}G in this "determined-arm" allele, DA-TLC1, by 61 kcal/mol (-19%) compared to wild type. We tested DA-TLC1 for reconstituted activity and found it to be [~]5-fold more robust than wild-type TLC1, suggesting that the inverse-folding design indeed improved folding in vitro into a catalytically active conformation. We also tested if DA-TLC1 functions in vivo and found that it complements a tlc1{triangleup} strain, allowing cells to avoid senescence and maintain telomeres of nearly wild-type length. However, all inverse-designed RNAs that we tested had reduced abundance in vivo. In particular, inverse-designing nearly all of the Ku arm caused a profound reduction in telomerase RNA abundance in the cell and very short telomeres. Overall, these results show that inverse design of S. cerevisiae telomerase RNA increases activity in vitro, while reducing abundance in vivo. This study provides a biochemically and biologically tested approach to inverse-design RNAs using Mfold that could be useful for controlling RNA structure in basic research and biomedicine.

molecular biology↗

bioRxiv2023

The widespread influence of ZSWIM8 on microRNAs during mouse embryonic development

MicroRNAs (miRNAs) pair to sites in mRNAs to direct the degradation of these RNA transcripts. Conversely, certain RNA transcripts can direct the degradation of particular miRNAs. This target-directed miRNA degradation (TDMD) requires the ZSWIM8 E3 ubiquitin ligase. Here, we report the function of ZSWIM8 in the mouse embryo. Zswim8-/- embryos were smaller than their littermates and died near the time of birth. This highly penetrant perinatal lethality was apparently caused by a lung sacculation defect attributed to failed maturation of alveolar epithelial cells. Some mutant individuals also had heart ventricular septal defects. These developmental abnormalities were accompanied by aberrant accumulation of >50 miRNAs observed across 12 tissues, which often led to enhanced repression of their mRNA targets. These ZSWIM8-sensitive miRNAs were preferentially produced from genomic miRNA clusters, and in some cases, ZSWIM8 caused a switch in the dominant strand that accumulated from a miRNA hairpin--observations suggesting that TDMD provides a mechanism to uncouple co-produced miRNAs from each other. Overall, our findings indicate that the regulatory influence of TDMD in mammalian biology is widespread and posit the existence of many yet-unidentified transcripts that trigger miRNA degradation.

molecular biology↗

bioRxiv2023

Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies

Full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensity than conventionally-labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5 or 3 end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a [~]6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence and fluorescence correlation spectroscopy showed that [~]3-fold brightness difference is due to static quenching most likely by the oligo or solution in the ext. configuration. Spectral flow cytometry experiments using peripheral blood mononuclear cells show int. MuSIC probe-labeled antibodies (i) retained increased signal intensity while having no significant difference in the estimated % of CD8+ lymphocytes and (ii) labeled with Atto488, Atto647, and Atto488/647 combinations can be demultiplexed in triple-stained samples. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications where spectral detection is available.

molecular biology↗

bioRxiv2023

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

We recently identified glycoRNA--a previously undescribed glycoconjugate--which consists of RNAs modified with secretory N-glycans and presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. The sensitivity and robustness of rPAL provided the first evidence of a direct glycan-RNA linkage, and its flexibility will enable further characterization of glycoRNA biology.

molecular biology↗

bioRxiv2025

CoSiDeX: A hyperspectral fluorescent protein resource for highly multiplexed imaging

Fluorescent proteins (FPs) have revolutionized spatiotemporal observations in biology. Yet, the design of multiplexed assays remains constrained by limited spectral characterization and palette validation. Although over 1,000 FPs have been catalogued, systematic resources for characterizing their use in multiplexed approaches are lacking. Here we present a resource and methodology for selecting and decoding FPs in multiplexed imaging experiments. A library of forty-four FPs was built for rapid assembly into mammalian expression vectors and transposase-mediated integration. Hyperspectral imaging was performed for each FP and spectral space was characterized mathematically. To support experimental design and data interpretation, we developed the Cosine Similarity Decoder of XFP (CoSiDeX) toolbox, to predict spectrally resolvable FP palettes and decode and re-color hyperspectral images. Using this approach, we demonstrate live-cell imaging of 12 uniquely labeled clones. Our work offers a scalable platform for selecting optimal FP palettes for multiplex experiments, with broad utility across diverse biological systems and hyperspectral imaging techniques.

molecular biology↗

bioRxiv2026

Circadian clock control of translation fidelity through MetRS-mediated methionine misincorporation

Translation fidelity is generally viewed as a constitutive process that deteriorates under stress and aging. Here we show that the fidelity of amino acid incorporation is instead dynamically regulated by the circadian clock. In Neurospora crassa, methionine (Met) misincorporation into proteins exhibits robust daily rhythms, peaking at night coincident with elevated reactive oxygen species (ROS). Rhythmic Met misincorporation requires the circadian clock, the ERK-family MAPK MAK1, and MAK1-dependent phosphorylation of methionyl-tRNA synthetase (MetRS), linking circadian signaling to regulated mistranslation associated with oxidative stress resistance. Preventing MetRS phosphorylation abolishes rhythmic Met misincorporation, impairs growth, and increases sensitivity to oxidative stress, whereas a phosphomimetic MetRS mutant enhances oxidative stress survival. Proteome-wide analyses identified thousands of Met misincorporation events, including a rhythmic subset that oscillates independently of corresponding protein abundance, suggesting that mistranslation dynamically remodels proteome composition across the day. Together, these findings establish translation fidelity as a regulated circadian output and support a model in which the circadian clock temporally regulates mistranslation to enhance oxidative stress resilience. Significance StatementBiological clocks regulate translation termination fidelity, but whether they also control the accuracy of amino acid incorporation during protein synthesis was unknown. We show that the circadian clock drives rhythmic methionine misincorporation into proteins through ERK-family MAPK signaling and phosphorylation of methionyl-tRNA synthetase. Methionine misincorporation peaks during periods of elevated oxidative stress, and disrupting this regulation compromises oxidative stress survival, whereas constitutive activation enhances resistance. Together with previous work on translation termination fidelity, these findings reveal that biological clocks regulate multiple layers of translation fidelity and identify adaptive mistranslation as a mechanism that promotes cellular resilience.

molecular biology↗

bioRxiv2026

LiverDCP: A Disease-Cell-Protein Framework for Multi-scale Modeling of Disease Biology

Understanding how molecular interactions give rise to disease phenotypes across cellular contexts remains a central challenge in biomedical research. Here, we introduce a Disease-Cell-Protein (DCP) paradigm for modeling multi-scale disease biology, which jointly represents disease states, cellular composition, and protein interaction networks within a unified graph architecture. We instantiate this paradigm in the liver as LiverDCP by integrating LiverHomo, a harmonized single-cell atlas of liver diseases, with proteome-wide predicted protein-protein interactions to construct over 280 context-specific interactomes across diverse liver disease and cellular conditions. LiverDCP employs a multi-context representation learning strategy that enables joint training across hundreds of disease-cell environments, capturing shared interaction principles while preserving context-specific variation. LiverDCP incorporates pretrained protein sequence-derived features through a geometry-aware two-phase training scheme that preserves embedding structure while improving predictive performance. The resulting DCP protein embeddings reveal extensive rewiring of protein functional states across diseases, providing a transferable representation for downstream biomedical applications. Without GWAS supervision during representation learning, LiverDCP enables disease-risk gene classification and identifies cell types through which genetic risk may act. For therapeutic target discovery, LiverDCP recovers established Phase II+ MASH targets and prioritizes previously unrecognized candidates from the unannotated proteome, with 26 of the top 50 predictions showing independent PubMed evidence related to MASH biology. Context-specific interaction analysis further provides mechanistic hypotheses for less-characterized candidates. Together, these results establish DCP as a generalizable framework for connecting molecular interactions, cellular context, genetic risk, and therapeutic opportunities across complex diseases.

systems biology↗

bioRxiv2021

Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering

Developing a biologic medicine requires successful decision making at each step of selection, optimization, and/or combination of the right candidates at early research stages. Knowing the structural information and binding pattern between drug target and discovery candidates greatly increases the possibility of success. With the cryo-EM resolution revolution and rapid development of computational software, we have evaluated and integrated different tools in structural biology and the computation field and established a highly cost-effective platform, which allows us to obtain fast and accurate structural information for biologics projects with a close to 100% success rate and as fast as weeks turn-around time. Here we report four case studies selected from over 40 different protein structures and share how we integrate cryo-EM structure determination, computational structure modeling, and molecular dynamics simulation. With proper decision making and strategic planning, the platform allows us to obtain quality results within days to weeks, including sub-100 kDa complexes which are usually considered as a challenge due to their small size. Our utilization of this differential approach and use of multiple software packages, allows to manage priorities and resources to achieve goals quickly and efficiently. We demonstrate how to effectively overcome particle orientation bias by altering complex composition. In several of our examples, we use glycan density to facilitate interpretation of low-resolution 3D reconstruction and epitope mapping. Protein information plays an important role in our cryo-EM projects, especially in cases where we see significant challenges in obtaining high-resolution 3D maps.

molecular biology↗

bioRxiv2021

A Two-Component System FleS/FleR Regulates Multiple Virulence-Related Traits in Pseudomonas aeruginosa

Microorganisms commonly use two-component systems (TCSs) to detect specific environmental changes and respond accordingly for their own benefit. However, the regulatory mechanisms and physiological roles of a majority of TCSs are still elusive. In this study, we focused on a previously predicted TCS FleS/FleR in Pseudomonas aeruginosa to systematically investigate its regulation and physiological roles. Loss of fleS or fleR or both genes led to decreased biofilm formation and attenuated motility in PAO1, which could be restored by heterologously complementation of FleR but not FleS, confirming that the sensor kinase FleS and the response regulator FleR constitute a TCS pair. To determine the regulatory spectrum of this TCS, we conducted transcriptome sequencing and comparison between the wild-type strain and the fleR deletion mutant. The result showed that the TCS regulates about 440 genes including most of them are involved in the virulence-related pathways, e.g. siderophore biosynthesis, pyocyanin biosynthesis, type III/VI secretion systems, c-di-GMP metabolism, flagellar assembly etc. In addition to its roles in controlling biofilm formation and motility we have already shown, FleR was demonstrated to regulate the production of virulence factors such as pyocyanin and elastase, mediate stress response to SDS, and autoregulate its own expression. Moreover, EMSA assays revealed that FleR regulates flagellum biosynthesis genes flgBCDE, flgFGHIJKL, filC, which are essential for the bacterial motility, by directly interacting with their promoters. Taken together, these results expanded our understanding on the biological roles of FleS/FleR and provided new insights on its regulatory mechanisms.

molecular biology↗

bioRxiv2021

RPRD Proteins Control Transcription in Human Cells

The regulation of transcription is an essential process that allows the cell to respond to various internal and external signals. RNA Polymerase II (Pol II) activity is controlled by a number of factors which bind to the C-terminal domain (CTD) of its largest subunit, RPB1, and stimulate or suppress RNA synthesis. Here, we demonstrate that CTD-interacting proteins, RPRD2, RPRD1B and RPRD1A act as negative regulators of transcription and their levels inversely correlate with the accumulation of nascent and newly transcribed RNA in human cells. We show that the RPRD proteins form mutually exclusive complexes with Pol II to coordinate their roles in transcriptional control. Our data indicate that RPRD2 exerts the most substantial impact on transcription and has the potential to alter key biological processes including the cellular stress response and cell growth.

molecular biology↗

bioRxiv2021

Alpha-Gal Syndrome: Involvement of Amblyomma americanum α-D-galactosidase and β-1,4 Galactosyltransferase enzymes in α-gal metabolism

Alpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose--1,3-galactose (-gal) injected into humans from the lone star tick (Amblyomma americanum) bite. This study aims at the functional characterization of two tick enzymes, -D-galactosidase (ADGal) and -1,4 galactosyltransferase ({beta}-1,4GalT) in -gal metabolism. The ADGal enzyme cleaves terminal -galactose moieties from glycoproteins and glycolipids, whereas {beta}-1,4GalT transfers -galactose to a {beta}1,4 terminal linkage acceptor sugars: GlcNAc, Glc, and Xyl in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and {beta}-1,4GalT in Am. americanum to examine their functional role in -gal metabolism and AGS onset. Silencing of ADGal led to the significant down regulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in -gal levels in silenced tissues. However, there was no significant difference in the level of -gal in {beta}-1,4GalT silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal silenced salivary glands. These results provide an insight into the role of -D galactosidase & {beta}-1,4GalT in tick biology and the probable involvement in the onset of AGS.

molecular biology↗

bioRxiv2021

Growing Glycans in Rosetta: Accurate de novo glycan modeling, density fitting, and rational sequon design

Carbohydrates and glycoproteins modulate key biological functions. Computational approaches inform function to aid in carbohydrate structure prediction, structure determination, and design. However, experimental structure determination of sugar polymers is notoriously difficult as glycans can sample a wide range of low energy conformations, thus limiting the study of glycan-mediated molecular interactions. In this work, we expanded the RosettaCarbohydrate framework, developed and benchmarked effective tools for glycan modeling and design, and extended the Rosetta software suite to better aid in structural analysis and benchmarking tasks through the SimpleMetrics framework. We developed a glycan-modeling algorithm, GlycanTreeModeler, that computationally builds glycans layer-by-layer, using adaptive kernel density estimates (KDE) of common glycan conformations derived from data in the Protein Data Bank (PDB) and from quantum mechanics (QM) calculations. After a rigorous optimization of kinematic and energetic considerations to improve near-native sampling enrichment and decoy discrimination, GlycanTreeModeler was benchmarked on a test set of diverse glycan structures, or "trees". Structures predicted by GlycanTreeModeler agreed with native structures at high accuracy for both de novo modeling and experimental density-guided building. GlycanTreeModeler algorithms and associated tools were employed to design de novo glycan trees into a protein nanoparticle vaccine that are able to direct the immune response by shielding regions of the scaffold from antibody recognition. This work will inform glycoprotein model prediction, aid in both X-ray and electron microscopy density solutions and refinement, and help lead the way towards a new era of computational glycobiology.

molecular biology↗

bioRxiv2023

Comprehensive structural and interactome analysis reveals 1 novel interactions and protein binding sites in miR-675: a non-coding RNA critically involved in multiple diseases

miR-675 is a microRNA expressed from exon 1 of H19 long non-coding RNA. H19 lncRNA is temporally expressed in humans and atypical expression of miR-675 has been linked with several diseases and disorders. To execute its function inside the cell, miR-675 is folded into a particular conformation which aids in its interaction with several other biological molecules. However, the exact folding dynamics of miR-675 and its complete interaction map are currently unknown. Moreover, how H19 lncRNA and miR-675 crosstalk and modulate each others activities is also unclear. Detailed structural analysis of miR-675 in this study determines its conformation and identifies novel protein binding sites on miR-675 which can make it an excellent therapeutic target against numerous diseases. Mapping of the interactome identified some of known and unknown interactors of miR-675 which aid in expanding our repertoire of miR-675 involved pathways in the cell. This analysis also identified some of the previously unknown and yet to be characterised proteins as probable interactors of miR-675. Structural and base pair conservation analysis between H19 lncRNA and miR-675 results in structural transformations in miR-675 thus describing the earlier unknown mechanism of interaction between these two molecules. Comprehensively, this study details the conformation of miR-675, its interacting biological partners and explains its relationship with H19 lncRNA which can be interpreted to understand the role of miR-675 in the development and progression of various diseases.

molecular biology↗

bioRxiv2023

High-throughput, microscopy-based screening, and quantification of genetic elements

Synthetic biology relies on the screening and quantification of genetic components to assemble sophisticated gene circuits with specific functions. Microscopy is powerful tool for characterizing complex cellular phenotypes with increasing spatial and temporal resolution to library screening of genetic elements. Microscopy-based assays are powerful tools for characterizing cellular phenotypes with spatial and temporal resolution, and can be applied to large-scale samples for library screening of genetic elements. However, strategies for high-throughput microscopy experiments remain limited. Here, we present a high-throughput, microscopy-based platform that can simultaneously complete the preparation of an 8x12-well agarose pads plate, allowing for the screening of 96 independent strains or experimental conditions in a single experiment. Using this platform, we screened a library of natural intrinsic promoters from Pseudomonas aeruginosa and identified a small subset of robust promoters that drives stable levels of gene expression under varying growth conditions. Additionally, the platform allowed for single-cell measurement of genetic elements over time, enabling the identification of complex and dynamic phenotypes to map genotype in high-throughput. We expected that the platform could be employed to accelerate the identification and characterization of genetic elements in various biological systems, as well as to understand the relationship between cellular phenotypes and internal states, including genotypes and gene expression programs. Impact statementThe high-throughput microscopy-based platform, presented in this study, enables efficient screening of 96 independent strains or experimental conditions in a single experiment, facilitating the rapid identification of genetic elements with desirable features, thereby advancing synthetic biology. The robust promoters identified through this platform, which provide predictable and consistent control over gene expression under varying growth conditions, can be utilized as reliable tools to regulate gene expression in various biological applications, including synthetic biology, metabolic engineering, and gene therapy, where consistent system performance is required.

molecular biology↗

bioRxiv2024

Structure-based probe reveals the presence of large transthyretin aggregates in plasma of ATTR amyloidosis patients

ATTR amyloidosis is a relentlessly progressive disease caused by the misfolding and systemic accumulation of amyloidogenic transthyretin into amyloid fibrils. These fibrils cause diverse clinical phenotypes, mainly cardiomyopathy and/or polyneuropathy. Little is known about the aggregation of transthyretin during disease development and whether this has implications for diagnosis and treatment. Using the cryogenic electron microscopy structures of mature ATTR fibrils, we developed a peptide probe for fibril detection. With this probe, we have identified previously unknown aggregated transthyretin species in plasma of patients with ATTR amyloidosis. These species are large, non-native, and distinct from monomeric and tetrameric transthyretin. Observations from our study open many questions about the biology of ATTR amyloidosis and reveals a potential diagnostic and therapeutic target.

molecular biology↗

bioRxiv2024

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei

Many Plasmodium genes remain uncharacterised due to low genetic tractability. Previous large scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called PbHiT, which uses a single cloning step to generate targeting vectors with 100 bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide vector designs and sequences to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasites biology at scale.

molecular biology↗

bioRxiv2024

Profiling of yeast Saccharomyces cerevisiae mitochondrial AMPylome reveals a regulation of ATP synthase coupling trough subunit delta

The adenylation (AMPylation) of proteins as a posttranslational modification is used by bacteria during infection of host cells. These new virulence factors - AMPylases mainly belonging to the FIC domain containing proteins and constitute a potential drug target. Human FIC protein (HYPE) controls the activity of BiP chaperone under endoplasmic reticulum stress. No FIC family proteins have yet been identified in yeast Saccharomyces cerevisiae. The second family of AMPylases are SelO proteins which control the redox homeostasis in mitochondria and chloroplasts. We describe here the first global screening of AMPylated proteins in yeast S. cerevisiae mitochondrial proteome from wild type and SelO (Fmp40) lacking cells. Through quantitative mass-spectrometry-based proteomics, we identified a total of 169 AMPylated proteins in mitochondria while AMPylated peptides of 115 proteins were identified in fmp40{Delta} mitochondria, indicating on the presence of another, besides Fmp40, not yet identified AMPylase in yeast. We confirmed AMPylation of Atp1, Atp2, Atp3 and Atp16 subunits of mitochondrial ATP synthase by western blotting. Interestingly, we found AMPylation and phosphorylation of many residues, what indicates on the complex regulation of the ATP synthase activity. We confirmed the importance of one of such residues in Atp16, showing that its post-translational modification serves to regulate ATP synthase and OXPHOS coupling in both fermentative and respiratory growth conditions. This regulation serves to maintain the proper potential of the inner mitochondrial membrane, particularly under conditions of fermentative growth. This dataset represents the first library of AMPylated mitochondrial yeast proteins reported to date and supplements the AMPylome of human chronic lymphocytic leukemia cell line from human HYPE containing and HYPE lacking cells. The data represents a foundation for substrate specific investigations that can ultimately decipher the biological role of the AMPylation in the mitochondria.

molecular biology↗

bioRxiv2017

SLAM-ITseq: Sequencing cell type-specific transcriptomes without cell sorting

Cell type-specific transcriptome analysis is an essential tool in understanding biological processes but can be challenging due to the limits of microdissection or fluorescence-activated cell sorting (FACS). Here, we report a novel in vivo sequencing method, which captures the transcriptome of a specific type of cells in a tissue without prior cellular or molecular sorting. SLAM-ITseq provides an accurate snapshot of the transcriptional state in vivo.

molecular biology↗