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Results for “molecular biology”

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bioRxiv2024

Development and Characterization of Recombinant ADP-Ribose Binding Reagents that Allow Simultaneous Detection of Mono and Poly ADP-Ribose

ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADP-ribosylation. Recently we developed a set of recombinant antibody-like ADP-ribose binding proteins, using naturally occurring ADPR binding domains (ARBDs) that include macrodomains and WWE domains, that have been functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with two other species, the Fc for mouse and goat immunogloblulin. Characterization of the new reagents indicates that they can be detected in a species-dependent manner, recognize specific ADP-ribose moieties, and excitingly, can be used in various antibody-based assays by co-staining. The expansion of this tool will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.

molecular biology↗

bioRxiv2024

Systematic screen of RNA binding proteins that enhance circular RNA translation

Translatable circular RNAs (circRNAs) have emerged as a promising alternative to linear mRNA as new therapeutics due to its improved stability. The translation of circRNAs is mainly driving by internal ribosome entry site (IRES) or IRES-like elements, which is under regulation by various trans-acting RNA binding proteins (RBPs). Here we designed a cell-based system to systematically screen RBPs that enhance translation of circRNAs, and identified a total of 68 proteins as putative activators of noncanonical translation. These translation activators mainly involved in the functions of RNA processing, ribosomal biogenesis and translation initiation. Furthermore, we developed a machine learning algorithm to extract common sequence features of these activators, which predicted more potential RBPs with translation activator activities. The newly identified and predicted activators were subsequently demonstrated to promote the IRES-mediated circRNA translation in a context-dependent fashion. This investigation provides new insights to discover functions for IRES trans-acting factors and to expand the toolbox for engineered RBPs in RNA synthetic biology.

molecular biology↗

bioRxiv2018

Excised linear introns regulate growth in yeast

Spliceosomal introns are ubiquitous non-coding RNAs typically destined for rapid debranching and degradation. Here, we describe 34 excised Saccharomyces cerevisiae introns that, although rapidly degraded in log-phase growth, accumulate as linear RNAs under either saturated-growth conditions or other stresses that cause prolonged inhibition of TORC1, a key integrator of growth signaling. Introns that become stabilized remain associated with components of the spliceosome and differ from the other spliceosomal introns in having a short distance between their lariat branch point and 3' splice site, which is necessary and sufficient for their stabilization. Deletion of these unusual introns is disadvantageous in saturated conditions and causes aberrantly high growth rates of yeast chronically challenged with the TORC1 inhibitor rapamycin. Reintroduction of native or engineered stable introns suppresses this aberrant rapamycin response. Thus, excised introns function within the TOR growth-signaling network of S. cerevisiae, and more generally, excised spliceosomal introns can have biological functions.

molecular biology↗

bioRxiv2025

Yeast as a platform to dissect poly(ADP-ribose) polymerase function from Magnaporthe oryzae and evaluate PARP inhibitors

Poly(ADP-ribose) polymerases (PARPs) regulate genome maintenance through NAD+-dependent ADP-ribosylation, yet PARP function in fungi remains poorly defined. Here, we reconstituted the activity of the Magnaporthe oryzae PARP1 homolog (MoPARP1) in Saccharomyces cerevisiae, a genetically tractable organism that lacks endogenous PARP enzymes. Galactose-induced expression of MoPARP1 reduced yeast growth, whereas a catalytically inactive mutant showed no defect, indicating that the growth phenotype depends on PARP catalytic activity. In a multidrug transporter-deficient background, the PARP inhibitor 3-aminobenzamide and the clinically used PARP inhibitor olaparib rescued the growth of MoPARP1-expressing strains, establishing a framework for inhibitor testing in vivo. Finally, MoPARP1-GFP localized to the nucleus independent of catalytic activity, supporting correct targeting in this heterologous system. Together, these findings establish yeast as a platform to dissect fungal PARP biology and evaluate chemical inhibition.

molecular biology↗

bioRxiv2026

Genomic and Functional Insights into the Cluster V Mycobacteriophage ‘EniyanLRS’ and its therapeutically relevant LysB

Multidrug-resistant tuberculosis and the rising burden of nontuberculous mycobacterial diseases demand innovative treatment strategies. Mycobacteriophages and endolysins offer promising therapeutic potential due to their targeted bacterial cell wall-damaging activity, vast diversity, and lack of antibiotic cross-resistance. Here, we report the genomic and functional characterisation of a V Cluster mycobacteriophage, EniyanLRS, and its endolysins. Some genomic features: genome size (78.53 kbp), a relatively low GC content (56.9%), a long Tape Measure Protein gene (5.97 kbp), and a high number of tRNAs (24), suggest distinct adaptation and translational efficiency of our phage. Phenotypically, EniyanLRS exhibits a siphovirus morphology, lytic life cycle and infects drug-resistant Mycobacterium fortuitum. While its endolysin LysA, harbouring a lysozyme-chitinase-amidase domain, showed moderate lysozyme-like activity without significant antibacterial activity, LysB, an /{beta}-hydrolase, exhibited superior in vitro esterase activity and showed pronounced cell wall disruption in M. smegmatis and M. fortuitum, along with considerable antibiofilm activity (62.77% and 41.91% inhibition, respectively). Collectively, our analysis reveals key functional properties of EniyanLRS phage and its LysB, demonstrating their suitability as effective biological candidates for mycobacterial therapy.

Molecular Biology↗

bioRxiv2026

RNA-dependent RNA amplification as a strategy to perform tRNA sequencing on low-input samples

Transfer RNAs (tRNAs) are key molecules that deliver amino acids to the translating ribosome according to their cognate codon encoded in messenger RNA (mRNA). Due to the modified nature of tRNA nucleotides, accurate tRNA quantification can be tedious, especially when dealing with small sample inputs. Here, we took advantage of an RNA-dependent RNA amplification method using T7 polymerase to quantify tRNA abundance in low biological input. Our method, called T7 High-resolution original RNA (Thor)-tRNA-Seq, showed reproducible and quantitative measurement of low tRNA inputs. Thus, our Thor-tRNA-Seq is a robust and reliable approach for the quantification of tRNA in samples with small and precious biological material.

molecular biology↗

bioRxiv2024

A Novel Partitivirus Confers Dual Contradictory Effect to Its Host Fungus: Growth Attenuation and Virulence Enhancement

Mycoviruses represent potential role in the biocontrol approach due to their ability to reduce both virulence and vegetative growth in some phytopathogenic fungi. However, the mycoviruses enhancing the damage caused by these fungi remains poorly identified and characterized. In this study, a novel double-stranded RNA (dsRNA) fungal virus, tentatively named Sinodiscula camellicola partitivirus 1 (ScPV1), was identified in the phytopathogenic fungus Sinodiscula camellicola, isolated from tea leaves. ScPV1 possesses two genomic components of 1835 and 1697 bp, each containing an open reading frame (ORF) that encodes the putative RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively, as confirmed by mass spectrometry. Phylogenetic analysis of the amino acid sequences of the RdRps from ScPV1 and related mycoviruses placed ScPV1 within a newly proposed genus, Epsilonpartitivirus, of the family Partitiviridae. Viral purification via ultracentrifugation and transmission electron microscopy observations revealed that ScPV1 dsRNA genomes are encapsidated within virus particles approximately 31 nm in size, ranging from 24.9 to 36.8 nm, along with the RdRp protein in an unexpected size. Transfection experiments with purified virions exhibited transfectants with significant reduced growth rates while increased virulence, indicating that ScPV1 has two-sided effect on its host fungus. This finding represents a significant advancement in understanding the complex interactions between mycoviruses and their host fungi. Author summaryTo characterize new molecular and biological traits of partitiviruses will provide substantial contributions for a better understanding of mycoviruses, as partitiviruses are prevalent across a wide range of hosts, including plants, protists, and fungi. Here, we identified a novel partitivirus, tentatively named Sinodiscula camellicola partitivirus 1 (ScPV1), from a fungus infecting tea plant, marking the first report of a partitivirus from a phytopathogenic fungus infecting tea plant. ScPV1 is characterized by two dsRNA genomic components encapsidated in particles of varying sizes, along with an RNA-dependent RNA polymerase protein of an expected size and containing some unique amino acids, indicating its distinct molecular and morphological traits. Our biological tests on transfectants generated via protoplast transfection with purified virions have rigorously demonstrated that ScPV1 impairs vegetative growth while enhancing virulence in its fungal host. This finding represents the first instance of a mycovirus responsible for hypervirulence and exhibiting dual effects on a phytopathogenic fungus through virion-transfection, as well as the first case of a partitivirus conferring hypervirulence while promoting vegetative growth in a phytopathogenic fungus. We anticipate that these findings will significantly advance our understanding of the complex interactions between mycoviruses and their host fungi.

microbiology↗

bioRxiv2020

Adaptive value of circadian rhythms in High Arctic Svalbard ptarmigan

The arctic archipelago of Svalbard (74 to 81{degrees} North) experiences extended periods of uninterrupted daylight in summer and uninterrupted darkness in winter. Species native to Svalbard display no daily rhythms in behaviour or physiology during these seasons, leading to the view that circadian rhythms may be redundant in arctic environments [1, 2]. Nevertheless, seasonal changes in the physiology and behaviour of arctic species rely on photoperiodic synchronisation to the solar year. Since this phenomenon is generally circadian-based in temperate species, we investigated if this might be a preserved aspect of arctic temporal organisation. Here, we demonstrate the involvement of the circadian clock in the seasonal photoperiodic response of the Svalbard ptarmigan (Lagopus muta hyperborea), the worlds northernmost resident bird species. First, we show the persistence of rhythmic clock gene expression under constant conditions within the mediobasal hypothalamus and pars tuberalis, the key tissues in the seasonal neuroendocrine cascade. We then employ a "sliding skeleton photoperiod" protocol, revealing that the driving force behind seasonal biology of the Svalbard ptarmigan is rhythmic sensitivity to light, a feature that depends on a functioning circadian rhythm. Our results suggest that the unusual selective pressure of the Arctic relaxes the adaptive value of the circadian clock for organisation of daily activity patterns, whilst preserving its importance for seasonal synchronisation. Thus, our data simultaneously reconnects circadian rhythms to life in the Arctic and establishes a universal principle of evolutionary value for circadian rhythms in seasonal biology.

molecular biology↗

bioRxiv2022

Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

Probing epigenomic marks such as histone modifications at a single cell level in thousands of cells has been recently enabled by technologies such as scCUT&Tag. Here we developed a multimodal and optimized iteration of scCUT&Tag called nano-CT (for nano-CUT&Tag) that allows simultaneous probing of three epigenomic modalities at single-cell resolution, using nanobody-Tn5 fusion proteins. nano-CT is compatible with starting materials as low as 25 000 cells and has significantly higher resolution than scCUT&Tag, with a 16-fold increase in the number of fragments per cells. We used nano-CT to simultaneously profile chromatin accessibility, H3K27ac and H3K27me3 in a complex tissue - juvenile mouse brain. The obtained multimodal dataset allowed for discrimination of more cell types/states that scCUT&Tag, and inference of chromatin velocity between ATAC and H3K27ac in the oligodendrocyte (OL) lineage. In addition, we used nano-CT to deconvolute H3K27me3 repressive states and infer two sequential waves of H3K27me3 repression at distinct gene modules during OL lineage progression. Thus, given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in different biological systems at single cell level.

molecular biology↗

bioRxiv2022

Monitoring promoter activity by RNA editing based reporter

Traditional methods monitoring the promoter activity require the insertion of reporter protein (e.g. fluorescent protein) downstream of a targeted promoter. These approaches suffer from low sensitivity and potential interference to endogenous transcripts especially when the targeted transcript is a noncoding RNA. Here, we develop a mechanistically different reporter system to monitor promoter activity based on ribozyme processed ADAR engaging RNA directed editing (REDDIT). We show that REDDIT can be used to monitor the promoter activity of protein coding, long noncoding RNA, and microRNA (miRNA) genes. Furthermore, REDDIT reporter can also be adapted to use bioluminescence imaging to monitor the promoter activity, which is more suitable for in vivo live imaging. Finally, the reporter sensitivity may be further increased by the circularization of ADAR recruiting RNA. REDDIT provides a powerful platform that is promising in a variety of applications such as monitoring the promoter activity, cell lineage tracing, and processing and manipulating genetic information for synthetic biology applications.

molecular biology↗

bioRxiv2022

Gene Set Scoring on the Nearest Neighbor Graph (gssnng) for Single Cell RNA-seq (scRNA-seq)

Gene set scoring (or enrichment) is a common dimension reduction task in bioinformatics that can be focused on differences between groups or at the single sample level. Gene sets can represent biological functions, molecular pathways, cell identities, and more. Gene set scores are context dependent values that are useful for interpreting biological changes following experiments or perturbations. Single sample scoring produces a set of scores, one for each member of a group, which can be analyzed with statistical models that can include additional clinically important factors such as gender or age. However, the sparsity and technical noise of single cell expression measures create difficulties for these methods, which were originally designed for bulk expression profiling (microarrays, RNAseq). This can be greatly remedied by first applying a smoothing transformation that shares gene measure information within transcriptomic neighborhoods. In this work, we use the nearest neighbor graph of cells for matrix smoothing to produce high quality gene set scores on a per-cell, per-group, level which is useful for visualization and statistical analysis. Availability and implementationThe gssnng software is available using the python package index (PyPI) and works with Scanpy AnnData objects. It can be installed using pip install gssnng. More information and demo notebooks: See https://github.com/IlyaLab/gssnng

bioinformatics↗

bioRxiv2023

Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells

Fluorescence In Situ Hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. Here, we present an improved method for FISH probe production that yields high purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, Amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The Degree Of Labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO and Alexa dyes) was mostly greater than 90%, comparable to commercial probes. The ease and low cost of production allowed generation of probe-sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs for Polr2a (RNA polymerase II subunit 2a) and Gapdh, and of the long noncoding RNAs Malat1 and Neat1. Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in the Gabbr1 and Noc2l transcripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

molecular biology↗

bioRxiv2024

Optogenetic control of phosphate-responsive genes using single component fusion proteins in Saccharomyces cerevisiae

Blue light illumination can be detected by Light-Oxygen-Voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here, we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling (PHO) pathway in the budding yeast Saccharomyces cerevisiae, exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration, and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (Pi) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionalities they can deliver and biological questions that can be probed.

molecular biology↗

bioRxiv2025

GOALS: Gene Ontology Analysis with Layered Shells for Enhanced Functional Insight and Visualization

Gene Ontologies (GOs) are standardized descriptions of gene functions in terms of biological processes, molecular functions, and cellular components, capturing their Parent-Child relationships in a structured framework and advancing cancer biological modeling to provide consistent and meaningful insights into functional genomics analysis. The conventional GO hierarchical structure is defined by human curation experts, with levels determined by the shortest path to the root term. However, grouping GOs poses challenges due to the uneven distribution of gene members within GO terms and inconsistencies in the level of detail across terms at the same GO level. In this work, we introduce Gene Ontology Analysis using Layered Shells (GOALS), a novel tool that discretizes GOAs into optimal layers. GOALS creates scalable GO layers while maintaining a balanced number of genes across GOs in each layer. Unlike existing tools, the GOALS framework organizes GO terms using a bottom-up approach based on their co-membership network, discretizing GOs to achieve an exponential fit with GOs gene member size. Meanwhile, GOALS reveals clusters or supersets reflecting biological relevance by unsupervised clustering of GOs latent projections. In a case study on mouse natural killer (NK) cell development, GOALS identified distinct GO functional clusters with multi-GO layers to reveal multiple levels of detail from specific to abstract contexts to maximize signal discovery and uncover those signals associations with trajectory divergence. More importantly, GOALS enhances enrichment analysis by introducing additional GO stratification and latent GO map that enables more accurate classification of functional differences. GOALS offers a robust and innovative framework for exploring disordered GO clusters, mining GO activities, and analyzing potential GO-GO interplays. By addressing critical challenges in functional genomics, GOALS provides a powerful tool for advancing our understanding of cell heterogeneity and potentially uncovering actionable insights for therapeutic development.

bioinformatics↗

bioRxiv2018

Previously characterized Caenorhabditis elegans RNA interference-resistant mutants retain substantial silencing capacity

RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

molecular biology↗

bioRxiv2019

Gibbs Free Energy, a thermodynamic measure of protein-protein interactions, correlates with neurologic disability

Background: Modern network science has been used to reveal new and often fundamental aspects of brain network organization in physiological as well as pathological conditions. As a consequence, these discoveries, which relate to network hierarchy, hubs and network interactions, begun to change the paradigms of neurodegenerative disorders. We therefore explored the use of thermodynamics for protein-protein network interactions in Alzheimer disease (AD), Parkinson disease (PD), multiple sclerosis (MS), traumatic brain injury and epilepsy. Methods: To assess the validity of using network interactions in neurological disease, we investigated the relationship between network thermodynamics and molecular systems biology for these neurological disorders. In order to uncover whether there was a correlation between network organization and biological outcomes, we used publicly available RNA transcription data from individual patients with these neurological conditions, and correlated these molecular profiles with their respective individual disability scores. Results: We found a linear correlation (Pearson correlation of -0.828) between disease disability (a clinically validated measurement of a person's functional status), and Gibbs free energy (a thermodynamic measure of protein-protein interactions). In other words, we found an inverse relationship between disease entropy and thermodynamic energy. Interpretation: Because a larger degree of disability correlated with a larger negative drop in Gibbs free energy in a linear, disability-dependent fashion, it could be presumed that the progression of neuropathology such as is seen in Alzheimer Disease, could potentially be prevented by therapeutically correcting the changes Gibbs free energy.

biophysics↗

bioRxiv2015

Methods for distinguishing between protein-coding and long noncoding RNAs and the elusive biological purpose of translation of long noncoding RNAs

Long noncoding RNAs (lncRNAs) are a diverse class of RNAs with increasingly appreciated functions in vertebrates, yet much of their biology remains poorly understood. In particular, it is unclear to what extent the current catalog of over 10,000 distinct annotated lncRNAs is indeed devoid of genes coding for proteins. Here we review the available computational and experimental schemes for distinguishing between coding and noncoding transcripts and assess the conclusions from their recent genome-wide applications. We conclude that the model most consistent with available data is that a large number of mammalian lncRNAs undergo translation, but only a very small minority of such translation events result in stable and functional peptides. The outcome of the majority of the translation events and their potential biological purposes remain an intriguing topic for future investigation.

Molecular Biology↗

bioRxiv2020

hu.MAP 2.0: Integration of over 15,000 proteomic experiments builds a global compendium of human multiprotein assemblies

A general principle of biology is the self-assembly of proteins into functional complexes. Characterizing their composition is, therefore, required for our understanding of cellular functions. Unfortunately, we lack a comprehensive set of protein complexes for human cells. To address this gap, we developed a machine learning framework to identify protein complexes in over 15,000 mass spectrometry experiments which resulted in the identification of nearly 7,000 physical assemblies. We show our resource, hu.MAP 2.0, is more accurate and comprehensive than previous resources and gives rise to many new hypotheses, including for 274 completely uncharacterized proteins. Further, we identify 259 promiscuous proteins that participate in multiple complexes pointing to possible moonlighting roles. We have made hu.MAP 2.0 easily searchable in a web interface (http://humap2.proteincomplexes.org/), which will be a valuable resource for researchers across a broad range of interests including systems biology, structural biology, and molecular explanations of disease.

systems biology↗