Biology subjects

Hennig, J.

Publications and source records attributed to Hennig, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein during translation regulation

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical {pi}-{pi}-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). This provides first atomistic details towards understanding regulation of translation initiation that is mediated by the interplay of these two proteins with each other and RNA.

molecular biology↗

bioRxiv2022

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless

Unwinding RNA secondary structures by RNA helicases is essential for RNA metabolism. How the basic unwinding reaction of DExH-type helicases is regulated by their accessory domains is unresolved. Here, we combine structural and functional analyses to address this challenge for the prototypic DExH RNA helicase maleless (MLE) from Drosophila. We captured the helicase cycle of MLE with multiple structural snapshots. We discovered that initially, dsRBD2 flexibly samples substrate dsRNA and aligns it with the open helicase tunnel. Subsequently, dsRBD2 releases RNA and associates with the helicase core, leading to closure of the tunnel around ssRNA. Structure-based MLE mutations confirm the functional relevance of the structural model in cells. We propose a molecular model in which the dsRBD2 domain of MLE orchestrates large structural transitions that depend on substrate RNA but are independent of ATP. Our findings reveal the fundamental mechanics of dsRNA unwinding by DExH helicases with high general relevance for dosage compensation and specific implications for MLEs human orthologue DHX9/RHA mechanisms in disease.

molecular biology↗

bioRxiv2022

Tropomyosin 1-I/C co-ordinates kinesin-1 and dynein motors during oskar mRNA transport

Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In Drosophila, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar mRNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. Here, through in vitro reconstitution, we show that Tm1-I/C, a Tropomyosin-1 isoform, links kinesin-1 in an inactive state to DDBE-associated oskar mRNA. NMR spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing a tug-of-war between the opposite polarity motors until kinesin-1 is activated in the oocyte. Our work reveals a novel strategy ensuring sequential activity of microtubule motors.

biochemistry↗

bioRxiv2021

The immune Rysto receptor recognizes a broadly conserved feature of potyviral coat proteins

Potyviruses are the largest group of plant RNA viruses, causing significant losses in many crops. Among them, potato virus Y (PVY) is particularly important, and enhances the severity of infections by other viruses. The Rysto gene confers PVY resistance and encodes a TIR-NLR intracellular immune receptors that recognizes PVY coat protein (CP). To define a minimal CP fragment sensed by Rysto, we created a series of truncated CP variants and expressed these CP derivatives in Rysto transgenic plants. Deletions that affect the 149 amino acid CP core region lose the ability to trigger Rysto-dependent defence activation. Furthermore, point mutations in the amino acid residues Ser126, Arg157, and Asp201 of the highly conserved RNA-binding pocket of potyviral CP, reduce or abolish Rysto-dependent responses, demonstrating that appropriate folding of the CP core is required for Rysto-mediated recognition. Consistent with these data, we found Rysto recognises CPs of various viruses that share a similar core region, but not those lacking it. Finally, we demonstrated that Rysto provides immunity to plum pox virus and turnip mosaic virus, demonstrating its wide range of applications in disease-resistant crop engineering. In parallel, we showed that CP triggered Rysto activation is SAG101- but not PAD4- or SA-level dependent. Our findings shed new light on how R proteins can detect viruses by sensing highly conserved structural patterns.

plant biology↗

bioRxiv2021

Structural basis of PETISCO complex assembly during piRNA biogenesis in C. elegans

Piwi-interacting RNAs (piRNAs) constitute a class of small RNAs that bind PIWI proteins and are essential to repress transposable elements in the animal germline, thereby promoting genome stability and maintaining fertility. C. elegans piRNAs (21U RNAs) are transcribed individually from minigenes as precursors that require 5 and 3 processing. This process depends on the PETISCO complex, consisting of four proteins: IFE-3, TOFU-6, PID-3, and ERH-2. We employ biochemical and structural biology approaches to characterize the PETISCO architecture and its interaction with RNA, together with its effector proteins TOST-1 and PID-1. These two proteins define different PETISCO functions: PID-1 governs 21U processing whereas TOST-1 links PETISCO to an unknown process essential for early embryogenesis. Here, we show that PETISCO forms an octameric assembly with each subunit present in two copies. Determination of structures of the TOFU-6/PID-3 and PID-3/ERH-2 subcomplexes, supported by in vivo studies of subunit interaction mutants, allows us to propose a model for the formation of the TOFU-6/PID-3/ERH-2 core complex, and its functionality in germ cells and early embryos. Using NMR spectroscopy, we demonstrate that TOST-1 and PID-1 bind to a common surface on ERH-2, located opposite its PID-3 binding site, explaining how PETISCO can mediate different cellular roles.

biochemistry↗

bioRxiv2021

Mechanism of riboregulation of p62 protein oligomerisation by vault RNA1-1 in selective autophagy

Macroautophagy ensures the clearance of intracellular substrates ranging from single ubiquitinated proteins to large proteotoxic aggregates and defective organelles. The selective autophagy receptor p62 binds these targets and recruits them to double-membrane vesicles, which fuse with lysosomes to degrade their content. We recently uncovered that p62 function is riboregulated by the small non-coding vault RNA1-1. Here, we present detailed insight into the underlying mechanism. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within the RNA that mediates the specific p62 interaction. Our data define molecular determinants that govern mammalian autophagy via the p62-vault RNA1-1 riboregulatory pair.

molecular biology↗

bioRxiv2021

Arabidopsis annexin 5 controls plasma membrane properties in mature pollen grains

In Arabidopsis, a dry stigma surface enables a gradual hydration of pollen grains by a controlled release of water. Occasionally the grains may be exposed to extreme precipitations that cause rapid water influx, swelling and eventually lead to pollen membrane (PM) rupture. In metazoans, calcium- and phospholipids-binding proteins, referred to as annexins participate in repair of the plasma membrane damages. It remains unclear, however, how this process is conducted in plants. Here, we examined whether the plant annexin 5 (ANN5), the most abundant member of the annexin family in pollen, is involved in the restoration of PM integrity. We analyzed a cellular dynamics of ANN5 in the pollen grains undergoing in vitro and in vivo hydration. We observed a transient ANN5 association to PM during the in vitro hydration that did not occur in the pollen grains being hydrated on the stigma. To simulate a rainfall, we performed spraying of the pollinated stigma with deionized water that induced ANN5 accumulation at PM. Similarly, calcium or magnesium application affected PM properties and induced ANN5 recruitment to PM. Our data suggest a model, in which ANN5 is involved in the maintenance of membrane integrity in pollen grains exposed to osmotic or ionic imbalances.

plant biology↗

bioRxiv2021

Validation and classification of RNA binding proteins identified by mRNA interactome capture

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we employed a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RNA binding proteins. Half of the analysed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster End binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1s RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather non-specific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

biochemistry↗

bioRxiv2020

RNA regulates Glycolysis and Embryonic Stem Cell Differentiation via Enolase 1

Cells must coordinate their metabolism and fate trajectories (1, 2), but the underlying mechanisms are only beginning to be discovered. To understand why the glycolytic enzyme enolase 1 (ENO1) binds RNA (3-6), we studied this phenomenon in vitro, in human cells, and during mouse embryonic stem cell differentiation. We find specific cellular RNA ligands that inhibit ENO1s enzymatic activity in vitro. Increasing the concentration of these ligands in cultured cells inhibits glycolysis. We demonstrate that pluripotent stem cells expressing an ENO1 mutant that is hyper-inhibited by RNA are severely impaired in their glycolytic capacity and in endodermal differentiation, whereas cells with an RNA binding-deficient ENO1 mutant display disproportionately high endodermal marker expression. Our findings uncover ENO1 riboregulation as a novel form of metabolic control. They also describe an unprecedented mechanism involved in the regulation of stem cell differentiation. One Sentence SummaryRNA directly regulates enzyme activity to control metabolism and stem cell fate

molecular biology↗

bioRxiv2020

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. The suppression of the RNA is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins in the RNA suppression remains a long-standing open question. We characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex is flexible with the unoccupied nucleotides of the RNA functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, Brat and Pum with Nanos bind the RNA completely independently. In accordance with previous studies, showing that Brat can suppress hunchback mRNA independently and is distributed uniformly throughout the embryo, this suggests that hunchback mRNA suppression by Brat is functionally separate from the suppression by Pumilio and Nanos.

molecular biology↗

bioRxiv2020

RNA binding regulates TRIM25-mediated RIG-I ubiquitylation

TRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25's E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA binding have been described, neither the precise RNA binding site has been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. We also show that TRIM25 not only binds RNA in mammalian cells but that interfering with RNA binding has an effect on cellular RIG-I ubiquitination.

molecular biology↗

bioRxiv2020

Transcriptional regulation of the Nε-fructoselysine metabolism in Escherichia coli by global and substrate-specific cues

Thermally processed food is an important part of the human diet. Heat-treatment, however, promotes the formation of so-called Amadori rearrangement products (ARPs), such as fructoselysine. The gut microbiota including Escherichia coli can utilize these compounds as a nutrient source. While the degradation route for fructoselysine is well described, regulation of the corresponding pathway genes frlABCD remained poorly understood. Here we use bioinformatics combined with molecular and biochemical analyses and show that in E. coli, fructoselysine metabolism is tightly controlled at the transcriptional level. The global regulator Crp (CAP), as well as the alternative sigma factor {sigma}32 (RpoH) contribute to promoter activation at high cAMP-levels and heat stress, respectively. In addition, we identified and characterized a transcriptional regulator FrlR, encoded adjacent to frlABCD, as fructoselysine-6-phosphate specific roadblock repressor. Our study provides profound evidence that the interplay of global and substrate-specific regulation is a perfect adaptation strategy to efficiently utilize unusual substrates within the human gut environment. Abbreviated SummaryThermal food processing promotes the formation of Amadori rearrangement products (ARPs), such as fructoselysine. The gut microbiota including Escherichia coli can utilize these compounds as a nutrient source. We show that in E. coli, fructoselysine metabolism is tightly controlled at the transcriptional level by global and substrate-specific regulators. Their interplay is a perfect adaptation strategy to efficiently utilize fructoselysine within the human gut environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/904318v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@15bbc3borg.highwire.dtl.DTLVardef@1a2e3eforg.highwire.dtl.DTLVardef@12261c6org.highwire.dtl.DTLVardef@412c7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Spatial accumulation of salicylic acid is in effector-triggered immunity of potato against viruses regulated by RBOHD

While activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to restriction of pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels to reveal spatiotemporal regulation of immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes, spatiotemporal regulation is completely lost in SA-deficient line, while the others show different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during resistance response. In plants with silenced RBOHD, the functionality of resistance response is perturbed and virus spread is not arrested at the site of infection. RBOHD is required for spatial accumulation of SA, and conversely RBOHD is under transcriptional regulation of SA signaling. Using spatially resolved RNA-Seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

plant biology↗

bioRxiv2019

Mapping the living mouse brain neural architecture: strain specific patterns of brain structural and functional connectivity

Mapping the structural and functional brain connectivity fingerprints became an essential approach in neurology and experimental neuroscience because network properties can underlie behavioral phenotypes. In mouse models, revealing strain related patterns of brain wiring have a tremendous importance, since these animals are used to answer questions related to neurological or neuropsychiatric disorders. C57BL/6 and BALB/cJ inbred strains are primary \"genetic backgrounds\" for brain disease modelling and for testing therapeutic approaches. Nevertheless, extensive literature describes basal differences in the behavioral, neuroanatomical and neurochemical profiles of the two strains, which raises the question whether the observed effects are pathology specific or depend on the genetic background. Here we performed a systematic comparative exploration of brain structure and function of C57BL/6 and BALB/cJ mice via Magnetic Resonance Imaging (MRI). We combined voxel-based morphometry (VBM), diffusion MRI and high resolution fiber mapping (hrFM) and resting state functional MRI (rs-fMRI) and depicted brain-wide dissimilarities in the morphology and \"connectome\" features in the two strains. Particularly C57BL/6 animals show bigger and denser frontal cortical areas, cortico-striatal tracts and thalamic and midbrain pathways, and higher density of fibers in the genu and splenium of the corpus callosum. These features are fairly reflected in the functional connectograms that emphasize differences in \"hubness\", frontal cortical and basal forbrain connectivity. We demonstrate strongly divergent reward-aversion circuitry patterns and some variations of the default mode network features. Inter-hemispherical functional connectivity showed flexibility and adjustment regarding the structural patterns in a strain specific manner. We further provide high-resolution tractograms illustrating also inter-individual variability across inter-hemispherical callosal pathways in the BALB/cJ strain.

neuroscience↗

bioRxiv2019

Structure-based screening of binding affinities via small-angle X-ray scattering

Protein-protein and protein-ligand interactions can alter the scattering properties of participating molecules, and thus be quantified by solution small-angle X-ray scattering (SAXS). In such cases, scattering reveals structural details of the bound complex, number of species involved, and in principle strength of the interaction. However, determining binding affinities from SAXS-based titrations is not yet an established procedure with well-defined performance expectations. We thus used periplasmic binding proteins and in particular histidine-binding protein as a standard reference, then examined precision and accuracy of affinity prediction at multiple concentrations and exposure times. By analyzing several structural and comparative scattering metrics, we found that the volatility of ratio between titrated scattering curves and a common reference most reliably quantifies ligand-triggered changes. This ratio permits the determination of affinities at low signal-to-noise ratios and without pre-determining the complex scattering, demonstrating that SAXS-based ligand screening is a promising alternative biophysical method for drug discovery pipelines.\n\nSIGNIFICANCESolution X-ray scattering can be used to screen a set of biomolecular interactions, which yields quantitative information on both structural changes and dissociation constants between binding partners. However, no common benchmarks yet exist for the application of SAXS within drug discovery workflows. Thus, investigations into its performance limitations are currently needed to make SAXS a reliable source for high-throughput screening. This study establishes a generalizable protocol based on protein-ligand interactions, and demonstrates its reproducibility across several beamline setups. In the simplest case, the micromolar binding affinities can be determined directly from measured intensities without knowledge of the molecular structure, with material consumption that is competitive with other biophysical screening techniques.

biophysics↗

bioRxiv2018

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

biochemistry↗

bioRxiv2018

Extreme resistance to Potato Virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor

Potato virus Y (PVY) is a major potato pathogen that causes annual losses of billions of dollars. Control of its transmission requires extensive use of environmentally damaging insecticides. Rysto confers extreme resistance (ER) to PVY and is a valuable trait in resistance breeding programs. We isolated Rysto using Resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences Single-Molecule Real Time Sequencing). Rysto encodes a nucleotide binding-leucine rich repeat (NLR) protein with an N-terminal TIR domain, and is sufficient for PVY perception and extreme resistance in transgenic potato plants. We investigated the requirements for Rysto-dependent extreme resistance, and showed that Rysto function is temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops.

plant biology↗

bioRxiv2018

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation

BackgroundPollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level.\n\nResultsHere, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis.\n\nConclusionsOur findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and embryo development.

plant biology↗