681 research outputs found
An evolutionarily conserved bimodular domain anchors ZC3HC1 and its yeast homologue Pml39p to the nuclear basket
No full textThe proteins ZC3HC1 and TPR are structural components of the nuclear basket (NB), a fibrillar structure attached to the nucleoplasmic side of the nuclear pore complex (NPC). ZC3HC1 initially binds to the NB in a TPR-dependent manner and can subsequently recruit additional TPR polypeptides to this structure. Here, we examined the molecular properties of ZC3HC1 that enable its initial binding to the NB and TPR. We report the identification and definition of a nuclear basket-interaction domain (NuBaID) of HsZC3HC1 that comprises two similarly built modules, both essential for binding the NB-resident TPR. We show that such a bimodular construction is evolutionarily conserved, which we further investigated in Dictyostelium discoideum and Saccharomyces cerevisiae. Presenting ScPml39p as the ZC3HC1 homologue in budding yeast, we show that the bimodular NuBaID of Pml39p is essential for binding to the yeast NB and its TPR homologues ScMlp1p and ScMlp2p, and we further demonstrate that Pml39p enables linkage between subpopulations of Mlp1p. We eventually delineate the common NuBaID of the human, amoebic, and yeast homologue as the defining structural entity of a unique protein not found in all but likely present in most taxa of the eukaryotic realm
Esteróis e triterpenos isolados de espécies de Ganoderma Karsten e sua atividade antimicrobiana
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em QuímicaGanoderma Karsten é um gênero de fungo pertencente a famíli
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Post-transcriptional regulation of gene expression by the DExD/H-box protein Dhh1
No full textBy repressing translation and promoting mRNA decay, cells are able to modulate gene expression and respond swiftly to changing environmental signals and developmental cues. Although translation, storage and degradation of mRNAs are key steps in the post-transcriptional control of gene expression, how mRNAs transit between these processes remains poorly understood. During my thesis I functionally characterized the DExD/H box ATPase Dhh1, a critical regulator of the cytoplasmic fate of mRNAs. Using mRNA tethering experiments in yeast, I showed that Dhh1 is sufficient to move an mRNA from an active state to translational repression. In actively dividing cells, translational repression is followed by mRNA decay, however, deleting components of the 5' to 3' decay pathway uncoupled these processes. Interestingly, Dhh1's ability to inactivate an mRNA coincided with its ability to move mRNAs into cytoplasmic processing bodies (P bodies). I also examined the role of ATP hydrolysis in Dhh1's ability to repress translation and activate mRNA decay. While Dhh1's ATPase activity is not essential for translational inhibition and mRNA decay in dhh1Δ cells, I found that ATP hydrolysis regulates P body dynamics and the release of Dhh1 from these RNA-protein granules. Surprisingly, I found that the presence of a wild-type copy of Dhh1 rescues the abnormal P-body localization of a Dhh1 ATPase-mutant. Additionally, the Dhh1 ATPase mutant no longer reduces mRNA and protein levels when tethered to an mRNA in the presence of a wild-type copy of Dhh1. My results place Dhh1 at the interface of translation and decay controlling whether an mRNA is translated, stored or decayed
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Development of a Novel Assay to Monitor Nuclear Pore Complex in Saccharomyces cerevisiae
No full textEukaryotic cells can be distinguished from prokaryotic cells because they posses membrane bound organelles. The presence of organelles in cells allowed cellular processes to be isolated into compartments, thus allowing additional levels of regulation to be applied to simple cellular processes. One of these membrane bound organelles, the nucleus, functions to isolate the cell's DNA from the cytoplasm. Large aqueous pores span the nuclear envelope and determines which molecules can enter and exit the nucleus. These channels are called Nuclear Pore Complexes (NPCs). The NPC is made up of a central core that spans the NE, a nuclear basket structure, fibers that extend into the cytoplasm, and is composed of only ~33 different proteins called Nucleoporins. The NPC exhibits an eight-fold rotation symmetry around the plane of the NE, and nucleoporins are present in eight, or multiples of eight, copies. Although the structure and composition of the NPC is well characterized, the process in which NPCs are inserted into the intact NE of yeast is unknown. In order to learn more about NPC assembly into the intact NE, I developed an assay to monitor the distribution of old and new nucleoporins in live S. cerevisiae cells. I used the photoconvertable fluorescent protein Dendra to examine a report that new NPC assembly occurs exclusively in daughter buds while old NPCs remain with mother cells. We examined two different Nups and observed new pore formation in both mother and daughter cells, additionally we determined that old pores are inherited by both mother and the daughter cells. We hypothesized that the differences in our observations from the previous report was due to differences experimental technique. To begin to understand the early events of NPC assembly, we first determined which proteins interact with the essential transmembrane Nup Ndc1. We then constructed conditional mutants of different combinations of these proteins and determined that Nup59 is functionally redundant with the combined element of Pom34 and Pom152. We show that these conditional mutants are not viable and result in mislocalization of core and cytoplasmic Nups. We use the Dendra assay to show that depletion of these elements result in a reversible defect in NPC assembly. Finally, we examine the role of Nup157 and Nup170 in NPC assembly. A nup157∆ strain conditionally expressing Nup170 results in mislocalization of core and cytoplasmic reporter Nups, but not nuclear Nups. This strain also has a reduced number of NPCs by EM in non-permissive conditions. Cells depleted of Nup170 and Nup157 were examined with the Dendra assay and new protein accumulated in the cytoplasm suggesting a block in NPC assembly. This block is overcome by reintroduction of Nup170. The work presented here represents a new way to study NPC assembly and reveals a few early events in NPC assembly
A role for Gle1, a regulator of DEAD-box RNA helicases, at centrosomes and basal bodies
No full textControl of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer's vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function
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The Dynamic Organization of the Yeast Genome
No full textRegardless of size, shape, or function, all cells must rapidly respond to a changing environment, especially in adverse conditions. Various environmental stresses including heat shock, osmotic stress, and nutrient starvation consequently induce dramatic changes in the molecular composition of cellular machinery. Importantly, the cell's adjustment to a new homeostasis is accomplished through a variety of mechanisms, but most predominantly through the regulation of gene expression which is normally thought to function through the interplay of specific transcriptional repressors and activators. Recent evidence suggests, however, that more novel forms of transcriptional regulation may exist, including the repositioning of specific genes to distinct subdomains within the nucleus and through global changes in genome organization. Moreover, it has been hypothesized that such changes may function to supplement more traditional models of transcriptional regulation. Thus, upon environmental stress, cells may dramatically modify global transcriptional programs through a reorganization of their genome, facilitating a more rapid response to an adverse environment. The budding yeast Saccharomyces cerevisiae represents a remarkable model for elucidating how the organization of the genome and the repositioning of specific genes may function to regulate gene expression. In yeast, certain cellular stresses are known to stimulate the repositioning of genes within the nucleus and alter the global organization of the genome. Despite considerable research into the dynamic nature of genome organization, however, many questions remain as to the true function of gene positioning and the mechanism(s) by which cells establish specific organizational conformations of the genome.In the following dissertation, I first provide evidence on the function of repositioning a specific gene to the nuclear periphery by examining the consequences of improperly localizing the GAL locus of budding yeast. Next, I describe my attempts to discern the coordinated diffusion of co-regulated genes as well as my findings on the independent movement of loci positioned various distances apart on the same chromosome. Finally, I describe novel findings on the mechanism of chromatin mobility through experiments focused on understanding the biological determinants of intracellular diffusion. Together, my results suggest that chromatin mobility is, in part, actively driven, which may function to facilitate more rapid alterations in genome organization and thus more rapid regulation of gene expression.I first set out to understand the function of repositioning specific genes to distinct locations within the nucleus. In yeast, the GAL locus, comprised of GAL1, GAL7, and GAL10 and necessary for the metabolism of galactose, re-localizes from a central position of the nucleus when repressed to the nuclear periphery when transcriptionally active. To understand the function of this specific repositioning, I took advantage of mutants in different macromolecular complexes, namely the nuclear pore complex and the SAGA transcriptional complex, which both fail to properly localize the GAL locus. I demonstrate that the GAL locus is negatively regulated at the nuclear periphery as both mutants, when compared to wild- type, displayed massively upregulated transcription of GAL1 upon activation and a distinct lag in repression. Importantly, the increased induction kinetics of GAL1 mRNA is mirrored by increased expression levels of GAL1 protein, suggesting that the over-expressed GAL1 mRNA is functional and that export of the mRNA is not perturbed despite the mislocalization of the GAL locus. In my model, the GAL locus is positioned at the nuclear periphery to facilitate more rapid transcriptional repression when cells encounter their preferred carbon source, glucose.Co-regulated genes may localize to transcriptional complexes known as transcription factories, hypothesized to facilitate more rapid and efficient transcription through colocalization and sequestration of specific transcriptional machinery. Because the GAL locus is known to re-localize at or near nuclear pore complexes upon activation, which may provide a scaffold for such transcriptional machinery, I next sought to observe transcription-induced coordinated diffusion of the two GAL loci within diploid yeast. To do so, I utilized a microscope capable of tracking both uniquely-labeled loci simultaneously using a double-helical point spread function. Although I did not discern evidence of potential transcription factories, I did reveal insights into the flexibility of chromatin by analyzing intrachromosomal loci separated by known distances. Overall, my results suggest that coordinated mobility of distinct loci correlates directly with their distance apart in space, regardless if they are intra or interchromosomal.Finally, I explored the mechanism of chromatin mobility and, thereby, the driving force behind the re-organization of the genome by comparing the diffusion rates of chromatin and other macromolecules in distinct conditions. I began my investigation by starving cells of glucose and determining the effect on macromolecular mobility. Interestingly, both chromatin and mRNPs (messenger ribonucleoprotein particles) display a massive confinement in their mobility in such conditions. The reduction in mobility cannot be explained by a decrease in ATP, but can be replicated through a reduction of intracellular pH. I then show that both glucose starvation and reduction of intracellular pH induce a decrease in cell volume, increasing molecular crowding and reducing macromolecular mobility. Furthermore, I demonstrate a dependence of chromatin mobility on the cytoskeleton as simultaneous treatment with actin and microtubule depolymerizers specifically confines chromatin mobility. Lastly, by uncoupling metabolism and macromolecular mobility I propose that the preservation of an optimal cell volume is signaled independently from the energy status of the cell and discuss implications and future directions of this work
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Functional Characterization of the DEAD-box ATPase Dhh1 - a regulator of cytoplasmic mRNA fate
No full textRapid modulation of gene expression is critical for cells to respond to environmental challenges and initiate developmental programs. Cells employ a variety of mechanisms to achieve tight regulation of gene expression, including post-transcriptional control of active messenger RNA (mRNA) levels by inhibition of translation or by mRNA degradation. While mRNA production via transcription has been extensively characterized, our understanding of how mRNAs are partitioned between an actively translating state and an inactive state is limited. In this thesis I examine the role of a highly evolutionarily conserved protein, the DEAD-box ATPase Dhh1, in mRNA inactivation and turnover in S. cerevisiae. Previous work from our lab puts Dhh1 at the crossroads of mRNA fate. For example, artificially tethering Dhh1 to an mRNA is sufficient to trigger its degradation. In contrast, in cells compromised in the 5'-3' decay pathway, tethered Dhh1 can no longer direct degradation of the message, yet still possesses the ability to repress its translation. Moreover, ATPase activity of Dhh1 is critical for mRNA localization in the cell, as ATPase-deficient mutants of Dhh1 induce the constitutive formation of mRNA-protein (mRNP) foci known as Processing Bodies (PBs) - enigmatic cellular structures that can direct storage or degradation of mRNAs. However, mechanistically how Dhh1 functions in translation repression and mRNA decay, as well as its role in PB assembly has remained elusive. In the following dissertation, I further characterize Dhh1 activities in translation repression, mRNA degradation, and PB formation. Using the previously established tethering assay, I identified protein factors that are distinctly required for translational repression or mRNA decay by Dhh1. Furthermore, I discovered that a mutant of Dhh1 that cannot bind to ATP is unable to interact with the Ccr4-NOT deadenylase complex - a major intracellular machine involved in transcriptional regulation and mRNA turnover. Finally, I show that Not1, the major scaffold of the Ccr4-NOT complex, controls Dhh1 localization to PB foci. In summary, my work suggests that the ATPase activity of Dhh1 is regulated in vivo, and this regulation may ultimately determine the fate of an mRNA - whether it is actively translated in the cytoplasm, or delivered to Processing Bodies for degradation or storage
Specific interaction with the nuclear transporter importin α2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles
No full textImportin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPα proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMPαs control cellular development, we conducted a yeast two-hybrid screen for IMPα2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPα2 expression coincident with germ-line masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPα2-binding partner. PSPC1-IMPα2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay–based assay demonstrated direct, high-affinity PSPC1 binding to either IMPα2/IMPβ1 or IMPα6/IMPβ1. Coexpression of full-length PSPC1 and IMPα2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMPα2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPα2 isoform or small interfering RNA knockdown of IMPα2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPα2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs
Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes
No full textThe biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.This work was supported by National Science Foundation Grants MCB0349443 and 0920578 to J.M.Z. and L.L. and National Science Foundation Major Research Instrumentation Grant DBI-0722569 to D.B. and T.G. Finally, we acknowledge the use of the UCSF Chimera Package supported by National Institute of General Medical Sciences Grant P41-GM103311.https://www.molbiolcell.org/doi/10.1091/mbc.e13-02-009
HQ peptide labeling of KARMA baits relative to their position in the protein sequence
No full textSupplementary analyzed data from Manuscript:
TITLE: Maturation kinetics of a multiprotein complex revealed by metabolic labeling.
JOURNAL: CELL.
Article Type: Research Article
Authors: Evgeny Onischenko*, Elad Noor*, Jonas S. Fischer*, Ludovic Gillet, Matthias Wojtynek, Pascal Vallotton, Karsten Weis
*Equally Contributing Authors
Corresponding Authors: Evgeny Onischenko and Karsten Weis
Related to Figure S4; Table S4
HQ peptide labeling of KARMA baits relative to their position in the protein sequence.
Individual Plots: Dependence of high-quality precursor labelling upon their position in the polypeptide chain was analysed for all ten KARMA bait proteins across all labelling time points. Note that the linear regression analysis does not show significant dependence between a peptide's labelling and its position in the polypeptide chain
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