Biology subjects

Weis, K.

Publications and source records attributed to Weis, K..

4 recordsLinked to original sources

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↗