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bioRxiv2025

Task-Geometry Alignment: a design principle forparameter-free, accurate genomic search

Standard alignment-based DNA barcoding falters because rigid geometric assumptions clash with biological realities, particularly insertions/deletions (indels) and fragment-to-reference asymmetry. To address this, we propose Task-Geometry Alignment (TGA), a design principle that structurally aligns the algorithm with the intrinsic geometry of the biological task. We implement TGA in TGAO_SCPLOWLIGNC_SCPLOW, a parameter-free tool that tiles reference databases into windows, applies gap-robust syncmer sketching, and indexes them for approximate nearest neighbor search. Across five benchmarks encompassing COI, 16S, and ITS markers, TGAO_SCPLOWLIGNC_SCPLOW achieves 9% higher accuracy and up to 10x faster query speeds than the leading aligner. Crucially, ablation studies confirm this architectural tiling is the causal driver of performance, allowing the tool to automatically match expert-parameterized configurations on fragment identification tasks. AvailabilitySource code and data are available at https://github.com/JustinBooneLab/TGAlign

molecular biology↗

bioRxiv2022

The RNA binding proteins ZFP36L1 and ZFP36L2 modulate transcriptional and post-transcriptional genome-wide effects of glucocorticoids

Glucocorticoids (GCs) are one of the most used anti-inflammatory drugs worldwide. Despite their widespread use, our understanding of their post-transcriptional effects remains poorly understood. The tristetraprolin (TTP) RNA binding protein (RBP) family (ZFP36, ZFP36L1 and ZFP36L2) has been implicated in inflammation regulation via binding to AU-rich elements (ARE) in mRNAs, with TTP being implicated in GC modulation. We hypothesised that ZFP36L1 and ZFP36L2 are part of the GC pathway and tested this hypothesis in bronchial epithelium, which commonly encounters GC in vivo upon inhalation. Our data show that dexamethasone, a commonly used GC, modulated the levels, subcellular localisation and RNA binding of ZFP36L1/L2. Employing Frac-seq (subcellular fractionation and RNA-sequencing), we show that GC modulated distinct subsets of RNAs in a subcellular-dependent manner. In addition to their mostly known transcriptional effects (116 differentially expressed genes, DEGs), GCs modified the binding to monosomes of myriad mRNAs (83 differentially bound genes, DBGs). We also demonstrate that ZFP36L1/L2 modulated gene expression mainly at the total cytoplasmic and polyribosome binding levels. ZFP36L1/L2 down-regulation led to an increase in ARE-containing mRNAs and a pronounced modification of the effects of GC on gene expression. We observed a small overlap of genes modulated by GCs when comparing control and ZFP36L1/L2 knockdown cells, in a subcellular-dependent manner Our data also suggest a novel role for these RBPs and GCs in epithelial biology via regulation of mRNAs encoding proteins important for epithelial cell function including cellular structure. We believe that our data has further implications in how we investigate gene expression. We show the power of employing sub-cellular fractionation when analysing genome-wide effects for known transcriptional modulators such as GCs, as well as a tool to demonstrate the extent of the effect of RBPs on gene expression modulation beyond total RNA levels.

molecular biology↗

bioRxiv2023

Metagenomic detection and genetic characterization of human sapovirus among children in Nigeria

Using a metagenomic sequencing approach on stool samples from children with Acute Flaccid Paralysis (AFP), we describe the genetic diversity of Sapoviruses (SaVs) in children in Nigeria. We identified six near-complete genome sequences and two partial genome sequences. Multiple SaV genogroups and genotypes were detected, including GII (GII.4 and GII.8), GIV (GIV.1) and GI (GI.2 and GI.7). Sequence identity and phylogenetic analysis showed that the Nigerian SaVs were related to previously documented gastroenteritis outbreaks associated strains from China and Japan. Minor variations in the functional motifs of the nonstructural proteins NS3 and NS5 were confirmed in the Nigerian strains. To adequately understand the effect of such amino acid changes, a better understanding of the biological function of these proteins is vital. The identification of distinct SaVs reinforces the need for robust surveillance in acute gastroenteritis (AGE) and non-AGE cohorts to better understand SaVs genotype diversity, evolution, and its role in disease burden in Nigeria.

molecular biology↗

bioRxiv2024

Predicting RNA Structure and Dynamics with Deep Learning and Solution Scattering

Advanced deep learning and statistical methods can predict structural models for RNA molecules. However, RNAs are flexible, and it remains difficult to describe their macromolecular conformations in solutions where varying conditions can induce conformational changes. Small-angle X-ray scattering (SAXS) in solution is an efficient technique to validate structural predictions by comparing the experimental SAXS profile with those calculated from predicted structures. There are two main challenges in comparing SAXS profiles to RNA structures: the absence of cations essential for stability and charge neutralization in predicted structures and the inadequacy of a single structure to represent RNAs conformational plasticity. We introduce Solution Conformation Predictor for RNA (SCOPER) to address these challenges. This pipeline integrates kinematics-based conformational sampling with the innovative deep-learning model, IonNet, designed for predicting Mg2+ ion binding sites. Validated through benchmarking against fourteen experimental datasets, SCOPER significantly improved the quality of SAXS profile fits by including Mg2+ ions and sampling of conformational plasticity. We observe that an increased content of monovalent and bivalent ions leads to decreased RNA plasticity. Therefore, carefully adjusting the plasticity and ion density is crucial to avoid overfitting experimental SAXS data. SCOPER is an efficient tool for accurately validating the solution state of RNAs given an initial, sufficiently accurate structure and provides the corrected atomistic model, including ions. The method is available from: https://github.com/dina-lab3d/IonNet Our pipeline is available for use as a web server: https://bilbomd.bl1231.als.lbl.gov/ Statement of SignificanceUnderstanding the behavior of RNA in solution is critical for deciphering its biological functions, yet predicting its macromolecular conformation remains challenging. While advanced computational methods can predict RNA structures, their accuracy in solution is often limited by the absence of stabilizing ions and the failure to account for RNAs conformational flexibility. This study presents SCOPER, an innovative tool that addresses these challenges by integrating deep-learning-based ion binding site prediction with conformational sampling, offering a more reliable approach to validate and refine RNA structures against experimental SAXS data. We provide our source code and a web server that runs the pipeline.

molecular biology↗

bioRxiv2025

A biological PROTAC for α-synuclein.

The accumulation of misfolded and aggregation-prone proteins is the hallmark of neurodegenerative diseases such as Parkinsons disease and amyotrophic lateral sclerosis (ALS). -Synuclein aggregation drives Parkinsons disease pathology, and is a suitable target for selective protein clearance. Biological proteolysis targeting chimeras (bioPROTACs) aim to eliminate disease-causing intracellular proteins using host cell ubiquitination and degradation functions. Here, we describe a bioPROTAC comprising the E3 ubiquitin ligase domain of CHIP (carboxy terminus of Hsc70-interacting protein) fused to NbSyn87, a nanobody specific for -synuclein. Co-expression with -synuclein resulted in a significant decrease in the abundance or complete degradation of both wild-type and Parkinsons disease-associated mutant -synuclein (A53T, A53V, and G51D) dependent on cell type. The bioPROTAC also significantly reduced abundance of insoluble -synuclein aggregates. In contrast, CHIP-based bioPROTACs targeting superoxide dismutase 1 (SOD1) or LIM domain only 2 (LMO2) failed to degrade their targets and in some instances, increased target abundance due to stabilising interactions with the recognition domain. These findings demonstrate key parameters for consideration during BioPROTAC design including target half-life, bioPROTAC solubility, recognition domain binding affinity, molecular chaperone activity, and interdomain linker optimisation. This work demonstrates the use of CHIP-based bioPROTACs for therapeutic degradation of -synuclein in the synucleinopathies and provides insights for future targeted degrader development.

molecular biology↗

bioRxiv2025

Mitochondrial fusion controls the development of specialized mitochondrial structure and metabolism in rod photoreceptor cells

Mitochondria are dynamic organelles that undergo continuous morphological changes, yet exhibit unique, cell-type-specific structures. In rod photoreceptor cells of the retina, these structures include elongated mitochondria in the inner segments and a distinct, large, circular mitochondrion in each presynaptic terminal. The mechanisms underlying the establishment and maintenance of these specialized mitochondrial morphologies, along with their functional significance, are not well understood. Here, we investigate the roles of mitochondrial fusion proteins mitofusin 1 (MFN1) and mitofusin 2 (MFN2) in shaping these structures and maintaining photoreceptor cell health. Rod photoreceptor cell-specific ablation of MFN1 and MFN2 resulted in mitochondrial fragmentation by one month of age, suggesting that mitochondrial fusion is essential for the development of photoreceptor cell-specific mitochondrial structures. Notably, the layer structures of the retina examined by light microscopy appeared unaffected at this age. Following this time period, significant photoreceptor cell degeneration occurred by three months of age. Furthermore, we showed that impaired mitochondrial fusion perturbed the balance of proteins involved in glycolysis, oxidative phosphorylation (OXPHOS), and {beta}-oxidation, highlighting the critical role of mitochondrial fusion in ensuring the proper levels of proteins necessary for optimal energy metabolism. Additionally, we identified upregulation of cellular stress pathways such as endoplasmic reticulum (ER) stress and unfolded protein response (UPR), which arise in response to energy deprivation, and cytoprotective biosynthetic pathways mediated by CCAAT/enhancer-binding protein gamma (C/EBP{gamma}) and mammalian target of rapamycin complex 1 (mTORC1) signaling. In summary, our findings indicate that mitochondrial fusion through MFN1 and MFN2 is vital for the development of unique mitochondrial structures and proper energy production, underscoring the fundamental importance of mitochondrial dynamics in photoreceptor cell function and survival. Significance StatementsRod photoreceptor cells exhibit unique mitochondrial morphologies and high energy requirements. In this report, we examined how these unique mitochondrial structures are established and their biological significance. We identified that mitochondrial fusion is essential for the development of characteristic mitochondrial morphologies in rod photoreceptor cells. Furthermore, we demonstrated that impaired mitochondrial fusion disrupts the equilibrium of proteins associated with OXPHOS, glycolysis, and {beta}-oxidation, ultimately leading to an imbalance in cellular energy homeostasis. Our findings also revealed activation of cellular stress pathways, including ER stress and the UPR, which are likely triggered by energy depletion. Additionally, we identified activation of cytoprotective biosynthetic pathways that are engaged to preserve cellular homeostasis and function.

molecular biology↗

bioRxiv2019

SIRT6 as a transcriptional coactivator of GATA4 prevents doxorubicin cardiotoxicity independently of its deacylase activity

Activity dependent and independent functions for some enzymes are indispensable as significant biological regulators. Deacylase SIRT6 is well-known to improve stress resistance and promote lifespan extension through enzymatic activity-dependent gene silencing. However, whether and how SIRT6 non-enzymatically actives the transcriptional output hasnt been characterized. Here, we revealed SIRT6 as a coactivator of GATA4, an essential transcription factor for postnatal cardiomyocyte survival, promoting the expression of anti-apoptotic gene. Chemotherapeutic drug, doxorubicin (DOX), remarkably and rapidly decreased SIRT6 expression, leading to transcriptional repression of GATA4 and cardiomyocyte apoptosis. Interestingly, SIRT6 interacted with GATA4 yet enhanced GATA4 acetylation independent of its deacylase activity, by recruiting the acetyltransferase Tip60 to form a trimeric complex. Nonacyl-mimetic mutation of GATA4 thoroughly blocked its ability against DOX cardiotoxicity. Moreover, Sirt6 transgenic mice exhibited preserved cardiac function with attenuated GATA4 activity in response to DOX. Thus, our studies uncover a previously unrecognized role of SIRT6 in cardioprotection independently of deacylase activity, providing the molecular basis to prevent chemotherapeutic side effects.

molecular biology↗

bioRxiv2021

RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell-type

The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyse additions of uridines at the 3' end of RNAs such as the precursors of the tumour suppressor miRNA let-7, upon recruitment by the oncoprotein LIN28A. Consequently, let-7 family miRNAs are downregulated. Disruption of this TUT4/7 activity inhibits tumorigenesis and hence targeting TUT4/7 can be a potential anti-cancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell type specific manner. Subsequently, BCL2 which is a well-established target of miR-200c is upregulated. Therefore, TUT4/7 loss triggers deregulation of miRNA-mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be key when targeting TUT4/7 for cancer therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/451846v2_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@a18bdcorg.highwire.dtl.DTLVardef@1e5534forg.highwire.dtl.DTLVardef@c08e76org.highwire.dtl.DTLVardef@1c90423_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights TUT4/7 catalyse 3' uridylation of most miRNAs Loss of uridylated isomiRs leads to the gain of adenylated isomiRs TUT4/7-mediated miRNA deregulation and miRNA-mRNA interactions are specific to the cell line type Loss of TUT4/7-mediated RNA uridylation inhibits cancer cell function

molecular biology↗

bioRxiv2018

Organ-specific translation elongation rates measured by in vivo ribosome profiling

Protein synthesis and degradation are intricate biological processes involving more than a hundred proteins operating in a highly orches-trated fashion. Despite the progress, few options are available to access translation in live animals as the increase in animals complexity limits the repertoire of experimental tools that could be applied to observe and manipulate processes within animals body, organs, and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type specific translation elongation rates. It is based on a time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ among mouse organs and determined them to be 6.8, 5.2, and 4.4 amino acids per sec for liver, kidney, and skeletal muscle, respectively.\n\nSignificanceProtein synthesis is a vital biological process. Modern methods of genome editing enable generation of sophisticated animal models to study the regulation of protein synthesis in health end disease. However, the methods that could track various steps of translation at a gene level resolution in vivo are lacking, particularly in complex vertebrates, such as mice and rats. Here, we measured the translation elongation rate in several organs by delivering inhibitors specific to certain phases of translation directly through the mouse bloodstream. This study lays out a path for interrogating translation in animals in response to various genetic and dietary interventions.

molecular biology↗

bioRxiv2019

Developmental dynamics are a proxy for selective pressures on alternatively polyadenylated isoforms

Alternative polyadenylation (APA) leads to multiple transcripts from the same gene, yet their distinct functional attributes remain largely unknown. Here, we introduce APA-seq to detect the expression levels of APA isoforms from 3-end RNA-Seq data by exploiting both paired-end reads for gene isoform identification and quantification. Applying APA-seq, we detected the expression levels of APA isoforms from RNA-Seq data of single C. elegans embryos, and studied the patterns of 3 UTR isoform expression throughout embryogenesis. We found that global changes in APA usage demarcate developmental stages, suggesting a requirement for distinct 3 UTR isoforms throughout embryogenesis. We distinguished two classes of genes, depending upon the correlation between the temporal profiles of their isoforms: those with highly correlated isoforms (HCI) and those with lowly correlated isoforms (LCI) across time. This led us to hypothesize that variants produced with similar expression profiles may be the product of biological noise, while the LCI variants may be under tighter selection and consequently their distinct 3 UTR isoforms are more likely to have functional consequences. Supporting this notion, we found that LCI genes have significantly more miRNA binding sites, more correlated expression profiles with those of their targeting miRNAs and a relative lack of correspondence between their transcription and protein abundances. Collectively, our results suggest that a lack of coherence among the regulation of 3 UTR isoforms is a proxy for selective pressures acting upon APA usage and consequently for their functional relevance.

molecular biology↗

bioRxiv2020

A simple technique to classify diffraction data from dynamic proteins according to individual polymorphs

One often observes small but measurable differences in diffraction data measured from different crystals of a single protein. These differences might reflect structural differences in the protein and potentially reflect the natural dynamism of the molecule in solution. Partitioning these mixed-state data into single-state clusters is a critical step to extract information about the dynamic behavior of proteins from hundreds or thousands of single-crystal data sets. Mixed-state data can be obtained deliberately (through intentional perturbation) or inadvertently (while attempting to measure highly redundant single-crystal data). State changes may be expressed as changes in morphology, so that a subset of the polystates may be observed as polymorphs. After mixed-state data are deliberately or inadvertently measured, the challenge is to sort the data into clusters that may represent relevant biological polystates. Here we address this problem using a simple multi-factor clustering approach that classifies each data set using independent observables in order to assign each data set to the correct location in conformation space. We illustrate this method using two independent observables (unit cell constants and intensities) to cluster mixed-state data from chymotrypsinogen (ChTg) crystals. We observe that the data populate an arc of the reaction trajectory as ChTg is converted into chymotrypsin.

molecular biology↗

bioRxiv2023

Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA

Cytosine deaminases have important uses in the detection of epigenetic modifications and in genome editing. However, the range of applications of deaminases is limited by a small number of well characterized enzymes. To expand the toolkit of deaminases, we developed an in-vitro approach that bypasses a major hurdle with their severe toxicity in expression hosts. We systematically assayed the activity of 175 putative cytosine deaminases on an unprecedented variety of substrates with epigenetically relevant base modifications. We found enzymes with high activity on double- and single-stranded DNA in various sequence contexts including novel CpG-specific deaminases, as well as enzymes without sequence preference. We also report, for the first time, enzymes that do not deaminate modified cytosines. The remarkable diversity of cytosine deaminases opens new avenues for biotechnological and medical applications. Using a newly discovered non-specific, modification-sensitive double-stranded DNA deaminase, we developed a nondestructive single-enzyme 5-methylctyosine sequencing (SEM-seq) method. SEM-seq enables accurate, high-coverage, base-resolution methylome mapping of scarce biological material including clinically relevant cell-free DNA (cfDNA) and single-cell equivalent 10 pg input DNA. Using SEM-seq, we generated highly reproducible base-resolution 5mC maps, accounting for nearly 80% of CpG islands for a low input human cfDNA sample offering valuable information for identifying potential biomarkers for detection of early-stage cancer and other diseases. This streamlined protocol will enable robust, high-throughput, high-coverage epigenome profiling of challenging samples in research and clinical settings.

molecular biology↗

bioRxiv2024

Potent and Selective SETDB1 Covalent Negative Allosteric Modulator Reduces Methyltransferase Activity in Cells

A promising drug target, SETDB1, is a dual Kme reader and methyltransferase, which has been implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identified a low micromolar small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization led to the discovery of UNC10013, the first covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a kinact/KI of 1.0 x 106 M-1s-1 and demonstrated proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation was observed after treatment with UNC10013. Therefore, UNC10013 is a potent, selective and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/615363v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@6db8e2org.highwire.dtl.DTLVardef@1852ee3org.highwire.dtl.DTLVardef@c95bb6org.highwire.dtl.DTLVardef@1a43951_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

EPB41L4A-AS1 long noncoding RNA acts in both cis- and trans-acting transcriptional regulation and controls nucleolar biology

Mammalian genomes are pervasively transcribed into long noncoding RNAs (lncRNAs), whose functions and modes of action remain poorly understood. EPB41L4A-AS1 is an evolutionary conserved, broadly and highly expressed lncRNA that produces the H/ACA snoRNA SNORA13 from one of its introns. We studied the consequences of EPB41L4A-AS1 perturbation in breast cancer cells and found that it acts both in cis, to enhance transcription of the proximal EPB41L4A gene and additional genes in its two flanking topologically associated domains, and in trans by broadly regulating gene expression, including expression of snoRNAs, transcription of genes involved in nucleolar biology and the distribution of nucleolar proteins. These effects are phenocopied by the loss of SUB1, an interactor of EPB41L4A-AS1, and are observed following transient perturbations of EPB41L4A-AS1 that do not affect steady-state SNORA13 levels or the rRNA modification it helps install. Exogenous expression of the full-length EPB41L4A-AS1 locus but not SNORA13 expression can rescue the trans-acting transcriptional effects of its perturbation. The EPB41L4A-AS1 gene is thus a versatile locus producing RNA molecules acting on multiple levels for key cellular functions.

molecular biology↗

bioRxiv2021

Beyond fish eDNA metabarcoding: Field replicates disproportionately improve the detection of stream associated vertebrate species

Fast, reliable, and comprehensive biodiversity monitoring data are needed for environmental decision making and management. Recent work on fish environmental DNA (eDNA) metabarcoding shows that aquatic diversity can be captured fast, reliably, and non-invasively at moderate costs. Because water in a catchment flows to the lowest point in the landscape, often a stream, it can often collect traces of terrestrial species via surface or subsurface runoff along its way or when specimens come into direct contact with water (e.g., for drinking purposes). Thus, fish eDNA metabarcoding data can provide information on fish but also on other vertebrate species that live in riparian habitats. This additional data may offer a much more comprehensive approach for assessing vertebrate diversity at no additional costs. Studies on how the sampling strategy affects species detection especially of stream-associated communities, however, are scarce. We therefore performed an analysis on the effects of biological replication on both fish as well as (semi-)terrestrial species detection. Along a 2 km stretch of the river Mulde (Germany), we collected 18 1-L water samples and analyzed the relation of detected species richness and quantity of biological replicates taken. We detected 58 vertebrate species, of which 25 were fish and lamprey, 18 mammals, and 15 birds, which account for 50%, 24%, and 7% of all native species to the German federal state of Saxony-Anhalt. However, while increasing the number of biological replicates resulted in only 25% more detected fish and lamprey species, mammal, and bird species richness increased disproportionately by 69% and 84%, respectively. Contrary, PCR replicates showed little stochasticity. We thus emphasize to increase the number of biological replicates when the aim is to improve general species detections. This holds especially true, when the focus is on rare aquatic taxa or on (semi-)terrestrial species, the so-called bycatch. As a clear advantage, this information can be obtained without any additional sampling or laboratory effort when the sampling strategy is chosen carefully. With the increased use of eDNA metabarcoding as part of national fish bioassessment and monitoring programs, the complimentary information provided on bycatch can be used for biodiversity monitoring and conservation on a much broader scale.

molecular biology↗

bioRxiv2024

Uncoupling the functional roles of Coronavirus Nsp1

When host cells are infected with coronaviruses, the first viral protein produced is Nsp1. This protein inhibits host protein synthesis and induces host mRNA degradation to enhance viral proliferation. Despite its critical role, the mechanism by which Nsp1 mediates cellular mRNA degradation remains unclear. In this study, we use cell-free translation to address how the host mRNA stability is regulated by Nsp1. We reveal that SARS-CoV-2 Nsp1 binding to the ribosome is enough to trigger mRNA degradation independently of ribosome collisions or active translation. MERS-CoV Nsp1 inhibits translation without triggering degradation, highlighting mechanistic differences between the two Nsp1 counterparts. Nsp1 and viral mRNAs appear to co-evolve, rendering viral mRNAs immune to Nsp1-mediated degradation in SARS-CoV-2, MERS-CoV and Bat-Hp viruses. By providing new insights into the mode of action of Nsp1, our study helps to understand the biology of Nsp1 better and find new strategies for therapeutic targeting against coronaviral infections. SignificanceO_LICell-free assays allow the decoupling of Nsp1-mediated translation inhibition from RNA degradation. C_LIO_LINsp1 interaction with the ribosome is crucial for mRNA degradation, but active translation is not required. C_LIO_LISARS-CoV-2 Nsp1 induces mRNA degradation, while MERS-CoV Nsp1 inhibits translation without triggering degradation. C_LIO_LI5UTR-specific protection of viral mRNAs from Nsp1 indicates a co-evolutionary adaptation between the two features C_LI

molecular biology↗

bioRxiv2018

Kinetic Pathways of Topology Simplification by Type-II Topoisomerases in Knotted Supercoiled DNA

The topological state of covalently closed, double-stranded DNA is defined by the knot type K and the linking-number difference {Delta}Lk relative to unknotted relaxed DNA. DNA topoisomerases are essential enzymes that control the topology of DNA in all cells. In particular, type-II topoisomerases change both K and {Delta}Lk by a duplex-strand-passage mechanism and have been shown to simplify the topology of DNA to levels below thermal equilibrium at the expense of ATP hydrolysis. It remains a puzzle how small enzymes are able to preferentially select strand passages that result in topology simplification in much larger DNA molecules. Using numerical simulations, we consider the non-equilibrium dynamics of transitions between topological states (K, {Delta}Lk) in DNA induced by type-II topoisomerases. For a biological process that delivers DNA molecules in a given topological state (K,{Delta}Lk) at a constant rate we fully characterize the pathways of topology simplification by type-II topoisomerases in terms of stationary probability distributions and probability currents on the network of topological states (K,{Delta}Lk). In particular, we observe that type-II topoisomerase activity is significantly enhanced in DNA molecules that maintain a supercoiled state with constant torsional tension. This is relevant for bacterial cells in which torsional tension is maintained by enzyme-dependent homeostatic mechanisms such as DNA-gyrase activity.

molecular biology↗

bioRxiv2018

Towards best-practice approaches for CRISPR/Cas9 gene engineering

In recent years, CRISPR has evolved from \"the curious sequence of unknown biological function\" into a functional genome editing tool. The CRISPR/Cas9 technology is now delivering novel genetic models for fundamental research, drug screening, therapy development, rapid diagnostics and transcriptional modulation. Despite the apparent simplicity of the CRISPR/Cas9 system, the outcome of a genome editing experiment can be substantially impacted by technical parameters as well as biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome editing experiments.

molecular biology↗