Biology subjects

Schmidt, N.

Publications and source records attributed to Schmidt, N..

27 records · Page 2Linked to original sources

bioRxiv2021

Complete pan-plastome sequences enable high resolution phylogenetic classification of sugar beet and closely related crop wild relatives

BackgroundAs the major source of sugar in moderate climates, sugar-producing beets (Beta vulgaris subsp. vulgaris) have a high economic value. However, the low genetic diversity within cultivated beets requires introduction of new traits, for example to increase their tolerance and resistance attributes - traits that often reside in the crop wild relatives. For this, genetic information of wild beet relatives and their phylogenetic placements to each other are crucial. To answer this need, we sequenced and assembled the complete plastome sequences from a broad species spectrum across the beet genera Beta and Patellifolia, both embedded in the Betoideae (order Caryophyllales). This pan-plastome dataset was then used to determine the wild beet phylogeny in high-resolution. ResultsWe sequenced the plastomes of 18 closely related accessions representing 11 species of the Betoideae subfamily and provided high-quality plastome assemblies which represent an important resource for further studies of beet wild relatives and the diverse plant order Caryophyllales. Their assembly sizes range from 149,723 bp (Beta vulgaris subsp. vulgaris) to 152,816 bp (Beta nana), with most variability in the intergenic sequences. Combining plastome-derived phylogenies with read-based treatments based on mitochondrial information, we were able to suggest a unified and highly confident phylogenetic placement of the investigated Betoideae species. Our results show that the genus Beta can be divided into the two clearly separated sections Beta and Corollinae. Our analysis confirms the affiliation of B. nana with the other Corollinae species, and we argue against a separate placement in the Nanae section. Within the Patellifolia genus, the two diploid species Patellifolia procumbens and Patellifolia webbiana are, regarding the plastome sequences, genetically more similar to each other than to the tetraploid Patellifolia patellaris. Nevertheless, all three Patellifolia species are clearly separated. ConclusionIn conclusion, our wild beet plastome assemblies represent a new resource to understand the molecular base of the beet germplasm. Despite large differences on the phenotypic level, our pan-plastome dataset is highly conserved. For the first time in beets, our whole plastome sequences overcome the low sequence variation in individual genes and provide the molecular backbone for highly resolved beet phylogenomics. Hence, our plastome sequencing strategy can also guide genomic approaches to unravel other closely related taxa.

genomics↗

bioRxiv2021

Solving the Sample Size Problem for Resource Selection Analysis

O_LISample size sufficiency is a critical consideration for conducting Resource-Selection Analyses (RSAs) from GPS-based animal telemetry. Cited thresholds for sufficiency include a number of captured animals M [≥] 30 and as many relocations per animal N as possible. These thresholds render many RSA-based studies misleading if large sample sizes were truly insufficient, or unpublishable if small sample sizes were sufficient but failed to meet reviewer expectations. C_LIO_LIWe provide the first comprehensive solution for RSA sample size by deriving closed-form mathematical expressions for the number of animals M and the number of relocations per animal N required for model outputs to a given degree of precision. The sample sizes needed depend on just 2 biologically meaningful quantities: habitat selection strength and a novel measure of landscape complexity, which we define rigorously. The mathematical expressions are calculable for any environmental dataset at any spatial scale and are applicable to any study involving resource selection (including sessile organisms). We validate our analytical solutions using globally relevant empirical data including 5,678,623 GPS locations from 511 animals from 10 species (omnivores, carnivores, and herbivores living in boreal, temperate, and tropical forests, montane woodlands, swamps, and arctic tundra). C_LIO_LIOur analytic expressions show that the required M and N must decline with increasing selection strength and increasing landscape complexity, and this decline is insensitive to the definition of availability used in the analysis. Our results contradict conventional wisdom by demonstrating that the most biologically relevant effects on the utilization distribution (i.e. those landscape conditions with the greatest absolute magnitude of resource selection) can often be estimated with far fewer data than is commonly assumed. C_LIO_LIWe identify several critical steps in implementing these equations, including (i) a priori selection of expected model coefficients, and (ii) sampling intensity for background (absence/pseudo-absence) data within a given definition of availability. We show that random sampling of background data violates the underlying mathematics of RSA, leading to incorrect values for necessary M and N and potentially incorrect RSA model outputs. We argue that these equations should be a mandatory component for all future RSA studies. C_LI

ecology↗

bioRxiv2021

An Activator Locks Kv7.1 Channels Open by Electro-Mechanical Uncoupling and Allosterically Modulates its Pore with similarity to phosphatidylinositol-4,5-bisphosphat

Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with increased risk of arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we showed that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identified novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments. Summarizing, we provide structural and functional evidence for two independent Kv7.1 activating mechanisms by a single modulator.

biophysics↗

bioRxiv2021

Genome-Wide CRISPR Screening Identifies BRD9 as a Druggable Component of Interferon-Stimulated Gene Expression and Antiviral Activity

Transient interferon (IFN) induction of IFN-stimulated genes (ISGs) creates a formidable protective antiviral state. However, loss of appropriate control mechanisms can result in constitutive pathogenic ISG upregulation. Here, we used genome-wide loss-of-function screening to establish genes critical for IFN signaling, identifying all expected members of the JAK-STAT pathway and the previously unappreciated bromodomain-containing protein 9 (BRD9), a defining subunit of non-canonical BAF (ncBAF) chromatin remodeling complexes. Genetic knock-out or small-molecule mediated degradation of BRD9 limited IFN-induced expression of a subset of ISGs in multiple cell-types, and prevented IFN from exerting full antiviral activity against several RNA and DNA viruses. Mechanistically, BRD9 acts at the level of ISG transcription, exhibits a proximal association with STAT2 following IFN stimulation, and relies on its intact acetyl-binding bromodomain and unique ncBAF scaffolding function for activity. Given its druggability, BRD9 may be an attractive target for dampening constitutive ISG expression under certain pathogenic autoinflammatory conditions.

immunology↗

bioRxiv2020

Broken, silent, and in hiding: Tamed endogenous pararetroviruses escape elimination from the genome of sugar beet (Beta vulgaris)

Background and AimsEndogenous pararetroviruses (EPRVs) are widespread components of plant genomes that originated from episomal DNA viruses of the Caulimoviridae family. Due to fragmentation and rearrangements, most EPRVs have lost their ability to replicate through reverse transcription and to initiate viral infection. Similar to the closely related retrotransposons, extant EPRVs were retained and often amplified in plant genomes for several million years. Here, we characterize the complete genomic EPRV fraction of the crop sugar beet (Beta vulgaris, Amaranthaceae) to understand how they shaped the beet genome and to suggest explanations for their absent virulence. MethodsUsing next- and third-generation sequencing data and the genome assembly, we reconstructed full-length in silico representatives for the three host-specific EPRV families (beetEPRVs) in the B. vulgaris genome. Focusing on the canonical family beetEPRV3, we investigated its chromosomal localization, abundance, and distribution by fluorescent in situ and Southern hybridization. Key ResultsBeetEPRVs range between 7.5 and 10.7 kb (0.3 % of the B. vulgaris genome) and are heterogeneous in structure and sequence. Although all three beetEPRV families were assigned to the florendoviruses, they showed variably arranged protein-coding domains, different degrees of fragmentation, and preferences for diverse sequence contexts. We observed small RNAs that target beetEPRVs in a family-specific manner, indicating stringent epigenetic suppression. We localized beetEPRV3 on all 18 sugar beet chromosomes, occurring preferentially in clusters and associated with heterochromatic, centromeric and intercalary satellite DNAs. BeetEPRV3 variants also exist in the genomes of related wild species, indicating an initial beetEPRV3 integration 13.4 to 7.2 million years ago. ConclusionsOur study in beet illustrates the variability of EPRV structure and sequence in a single host genome. Evidence of sequence fragmentation and epigenetic silencing imply possible plant strategies to cope with long-term persistence of EPRVs, including amplification, fixation in the heterochromatin, and containment of EPRV virulence.

plant biology↗

bioRxiv2020

A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells

SARS-CoV-2 infections pose a global threat to human health and an unprecedented research challenge. Among the most urgent tasks is obtaining a detailed understanding of the molecular interactions that facilitate viral replication or contribute to host defense mechanisms in infected cells. While SARS-CoV-2 co-opts cellular factors for viral translation and genome replication, a comprehensive map of the host cell proteome in direct contact with viral RNA has not been elucidated. Here, we use RNA antisense purification and mass spectrometry (RAP-MS) to obtain an unbiased and quantitative picture of the human proteome that directly binds the SARS-CoV-2 RNA in infected human cells. We discover known host factors required for coronavirus replication, regulators of RNA metabolism and host defense pathways, along with dozens of potential drug targets among direct SARS-CoV-2 binders. We further integrate the SARS-CoV-2 RNA interactome with proteome dynamics induced by viral infection, linking interactome proteins to the emerging biology of SARS-CoV-2 infections. Validating RAP-MS, we show that CNBP, a regulator of proinflammatory cytokines, directly engages the SARS-CoV-2 RNA. Supporting the functional relevance of identified interactors, we show that the interferon-induced protein RYDEN suppresses SARS-CoV-2 ribosomal frameshifting and demonstrate that inhibition of SARS-CoV-2-bound proteins is sufficient to manipulate viral replication. The SARS-CoV-2 RNA interactome provides an unprecedented molecular perspective on SARS-CoV-2 infections and enables the systematic dissection of host dependency factors and host defense strategies, a crucial prerequisite for designing novel therapeutic strategies.

microbiology↗

bioRxiv2020

Cdk1-dependent destabilization of long astral microtubules is required for spindle orientation

The precise execution of mitotic spindle orientation in response to cell shape cues is important for tissue organization and development. The presence of astral microtubules extending from the centrosome towards the cell cortex is essential for this process, but little is understood about the contribution of astral microtubule dynamics to spindle positioning, or how astral microtubule dynamics are regulated spatiotemporally. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells transition from interphase to mitosis, but how Cdk1 activity specifically modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules to ensure spindle reorientation in response to cell shape. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus-ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus-ends in mitosis. This decreases the catastrophe frequency of astral microtubules, and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules must thus not only be present, but also dynamic to allow the spindle to reorient in response to cell shape, a state achieved by selective destabilization of long astral microtubules via Cdk1.

cell biology↗

bioRxiv2019

ESI mutagenesis: A one-step method for introducing point mutations into bacterial artificial chromosome transgenes

Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. While homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call ESI (Exogenous/Synthetic Intronization) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI-mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

molecular biology↗

bioRxiv2019

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Clathrin plays an important role to ensure mitotic spindle stability and efficient chromosome alignment, independently of its well-characterized functions in vesicle trafficking. While clathrin clearly localizes to the mitotic spindle and kinetochore-fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules have remained elusive. Here we show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the mitotic spindle. Structural analyses reveal that multiple clathrin-box motifs on GTSE1 interact directly with different clathrin adaptor interaction sites on CHC, in a manner structurally analogous to that which occurs between adaptor proteins and CHC near membranes. Specific disruption of this interaction in cells releases GTSE1 from spindles and causes defects in chromosome alignment. Surprisingly, this disruption causes destabilization of astral microtubules, but not kinetochore-microtubule attachments, and the resulting chromosome alignment defect is due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. Finally, we show that GTSE1 recruited to the spindle by clathrin stabilizes microtubules and promotes chromosome congression by inhibiting the activity of the microtubule depolymerase MCAK. This work thus uncovers a novel role of clathrin to stabilize non-kinetochore-fiber microtubules to support chromosome congression. This role is carried out via clathrin adaptor-type interactions of CHC with GTSE1, defining for the first time an important repurposing of this endocytic interaction mechanism during mitosis.

cell biology↗