Biology subjects

Weis, K.

Publications and source records attributed to Weis, K..

27 records · Page 2Linked to original sources

bioRxiv2022

Condensation of a nuclear mRNA export factor regulates mRNA transport during stress

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that upon glucose withdrawal the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and to control gene expression during stress. HighlightsO_LIThe nuclear poly(A)-binding protein Nab2 forms RNA-containing condensate-like structures upon glucose starvation and upon acute cellular depletion of the DEAD-box ATPase Dbp5 C_LIO_LIThe Nab2 multimerization interface but not the intrinsically disordered regions (IDRs) are essential for condensation in vitro and in vivo C_LIO_LIGlucose stress leads to poly(A) RNA retention in the nucleus, which is affected by the state of the Nab2 condensate C_LI

cell biology↗

bioRxiv2021

Characterization of RNA content in individual phase-separated coacervate microdroplets

Liquid-liquid phase separation or condensation is a form of macromolecular compartmentalization. Condensates formed by complex coacervation were hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of condensation in cells and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and how single condensates differ in their RNA composition. Therefore, we developed an approach to read the RNA content from single condensates using high-throughput sequencing. We find that RNAs which are enriched for specific sequence motifs efficiently accumulate in condensates. These motifs show high sequence similarity to short interspersed elements (SINEs). We observed similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

genomics↗

bioRxiv2021

Dynamic arrest and aging of biomolecular condensates are regulated by low-complexity domains, RNA and biochemical activity

Biomolecular condensates require suitable material properties to properly carry out their function. Yet, modulators that affect the material properties of condensates have remained largely unexplored. Here we apply Differential Dynamic Microscopy (DDM) to probe the material properties of an in vitro model of processing bodies (P-bodies) consisting of condensates formed by the DEAD-box ATPase Dhh1 in the presence of ATP and RNA. DDM allows us to measure the viscosity of liquid droplets and to distinguish between liquid-like and gel- or glass-like condensates. By applying this single-droplet technique we show that condensates within the same population exhibit a distribution of material properties, which can be drastically affected by several modulators such as the low-complexity domains (LCDs) of the protein, the protein/RNA ratio, the type of RNA as well as the enzymatic activity. In particular, we show that structured RNA leads to a large fraction of dynamically arrested condensates with respect to unstructured polyuridylic acid (polyU), emphasizing the role of RNA structure in condensate dynamics. We further demonstrate that the ageing of the condensates and the formation of gel or glass-like structures is reduced by promoting the enzymatic ATPase activity of Dhh1 and the rate of droplet formation and dissolution. Our study shows that not only the reversible formation and dissolution of condensates but also their material properties are regulated on several levels, and that biochemical activity and material turnover can be important to maintain fluid-like properties over time.

biophysics↗

bioRxiv2019

The RNA export factor Mex67 functions as a mobile nucleoporin

The RNA export factor Mex67 is essential for the transport of mRNA through the nuclear pore complex (NPC) in yeast, but the molecular mechanism of this export process remains poorly understood. Here, we use quantitative fluorescence micro-scopy techniques in live budding yeast cells to investigate how Mex67 facilitates mRNA export. We show that Mex67 exhibits little interaction with mRNA in the nucleus and localizes to the NPC independently of mRNA, occupying a set of binding sites offered by FG repeats in the NPC. The ATPase Dbp5, which is thought to remove Mex67 from transcripts, does not affect the interaction of Mex67 with the NPC. Strikingly, we find that the essential function of Mex67 is spatially restricted to the NPC since a fusion of Mex67 to the nucleoporin Nup116 rescues a deletion of MEX67. Thus, Mex67 functions as a mobile nuclear pore component, which receives mRNA export substrates in the central channel of the NPC to facilitate their translocation to the cytoplasm.

cell biology↗

bioRxiv2019

Lipid digestion and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae

The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant - though lower - level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics, to characterize the cells response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP to ATP ratio strongly increases within 20-30 seconds. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, bulk autophagy, a process that frees amino acids, as well as lipid degradation via {beta}-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and {beta}-oxidation as important energy providers during acute glucose starvation.

systems biology↗

bioRxiv2018

Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

biochemistry↗

bioRxiv2017

Quantitative imaging of chromatin decompaction in living cells

Chromatin organization is highly dynamic and regulates transcription. Upon transcriptional activation, chromatin is remodeled and referred to as \"open\", but quantitative and dynamic data of this decompaction process are lacking. Here, we have developed a quantitative high-resolution microscopy assay in living yeast cells to visualize and quantify chromatin dynamics using the GAL7-10-1 locus as a model system. Upon transcriptional activation of these three clustered genes, we detect an increase of the mean distance across this locus by >100 nm. This decompaction is linked to active transcription but is not sensitive to the histone deacetylase inhibitor trichostatin A or to deletion of the histone acetyl transferase Gcn5. By contrast, the deletion of SNF2 (encoding the ATPase of the SWI/SNF chromatin remodeling complex) or the deactivation of the histone chaperone complex FACT lead to a strongly reduced decompaction without significant effects on transcriptional induction. Our findings are consistent with nucleosome remodeling and eviction activities being major contributors to chromatin reorganization during transcription but also suggest that transcription can occur in the absence of detectable decompaction.

cell biology↗

bioRxiv2017

Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability

The cytoplasmic abundance of mRNAs is strictly controlled through a balance of production and degradation. Whereas the control of mRNA synthesis through transcription has been well characterized, less is known about the regulation of mRNA turnover, and a consensus model explaining the wide variations in mRNA decay rates remains elusive. Here, we combine non-invasive transcriptome-wide mRNA production and stability measurements with selective and acute perturbations to demonstrate that mRNA degradation is tightly coupled to the regulation of translation, and that a competition between translation initiation and mRNA decay -but not codon optimality or elongation- is the major determinant of mRNA stability in yeast. Our refined measurements also reveal a remarkably dynamic transcriptome with an average mRNA half-life of only 4.8 minutes - much shorter than previously thought. Furthermore, global mRNA destabilization by inhibition of translation initiation induces a dose-dependent formation of processing bodies in which mRNAs can decay over time.

genetics↗

bioRxiv2017

Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

The nuclear pore complex (NPC) is an 8-fold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ~30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure, however, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed Nuclear Rim Intensity Measurement or NuRIM, to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188 that their altered expression leads to significant changes in Nup stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.\n\nSignificanceThe nuclear pore complex (NPC) is one of the largest protein complexes in eukaryotes comprising over 500 nucleoporin subunits. The NPC is essential for transport of biomolecules across the nuclear envelope, however, due to its enormous size, it has been a challenge to characterize its molecular architecture. Herein, we have developed a novel, widely applicable imaging pipeline to determine the absolute nucleoporin abundances in native yeast NPCs. This reveals that the NPC composition dramatically differs between yeast and human despite an overall conservation of individual subunits. We also applied our imaging pipeline to examine yeast mutants revealing a remarkable compositional plasticity of NPCs. Our stoichiometry analyses provide an important resource for the generation of high-resolution structure models of the NPC.

cell biology↗