Generation of synthetic RNA-based thermosensors

2008 ◽  
Vol 389 (10) ◽  
Author(s):  
Torsten Waldminghaus ◽  
Jens Kortmann ◽  
Stefan Gesing ◽  
Franz Narberhaus
Keyword(s):  
Conformational Changes ◽  
Translational Control ◽  
Rational Design ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Structure Probing ◽  
Bacterial Rna ◽  
Computer Based ◽  
Shine Dalgarno Sequence

AbstractStructured RNAs with fundamental sensory and regulatory potential have been discovered in all kingdoms of life. Bacterial RNA thermometers are located in the 5′-untranslated region of certain heat shock and virulence genes. They regulate translation by masking the Shine-Dalgarno sequence in a temperature-dependent manner. To engineer RNA-based thermosensors, we used a combination of computer-based rational design andin vivoscreening. After only two rounds of selection, several RNA thermometers that are at least as efficient as natural thermometers were obtained. Structure probing experiments revealed temperature-dependent conformational changes in these translational control elements. Our study demonstrates that temperature-controlled RNA elements can be designed by a simple combined computational and experimental approach.

scholarly journals MAP6-F Is a Temperature Sensor That Directly Binds to and Protects Microtubules from Cold-induced Depolymerization

2012 ◽  
Vol 287 (42) ◽  
pp. 35127-35138 ◽  
Author(s):  
Christian Delphin ◽  
Denis Bouvier ◽  
Maxime Seggio ◽  
Emilie Couriol ◽  
Yasmina Saoudi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Temperature Sensor ◽  
Cold Sensitivity ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Microtubule Network ◽  
Wide Range ◽  
In Cells

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.

scholarly journals ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

2021 ◽  
Author(s):  
N. Amy Yewdall ◽  
Alain A. M. André ◽  
Merlijn H. I. van Haren ◽  
Frank H. T. Nelissen ◽  
Aafke Jonker ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Enzymatic Reaction ◽  
Atp Depletion ◽  
Gel Point ◽  
Dependent Manner ◽  
Biophysical Properties ◽  
Temperature Dependent ◽  
Quantitative Fluorescence

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus.

scholarly journals Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP

2016 ◽  
Vol 113 (27) ◽  
pp. 7545-7550 ◽  
Author(s):  
Rachel Ruoff ◽  
Olga Katsara ◽  
Victoria Kolupaeva
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Bone Development ◽  
Binding Protein ◽  
Initiation Factor ◽  
Start Codon ◽  
Dependent Manner ◽  
Fgf Signaling ◽  
Cell Type

Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type–dependent manner, with 4E-BP1 being a key player.

scholarly journals ACTIVATION LOOP PHOSPHORYLATION OF A NON-RD RECEPTOR KINASE INITIATES PLANT INNATE IMMUNE SIGNALING

2021 ◽  
Author(s):  
Kyle W Bender ◽  
Daniel Couto ◽  
Yasuhiro Kadota ◽  
Alberto P Macho ◽  
Jan Sklenar ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Stress Responses ◽  
Elongation Factor ◽  
Cytoplasmic Domain ◽  
Dependent Manner ◽  
Immune Signaling ◽  
Receptor Kinases

Receptor kinases (RKs) play fundamental roles in extracellular sensing to regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) function primarily as peptide receptors that regulate myriad aspects of plant development and response to external stimuli. Extensive phosphorylation of LRR-RK cytoplasmic domains is among the earliest detectable responses following ligand perception, and reciprocal transphosphorylation between a receptor and its co-receptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model RK, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in anti-bacterial immunity. These results reveal a non-catalytic role for the EFR cytoplasmic domain in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RK complexes with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor which could initiate signaling in a feed-forward fashion either allosterically or through driving the dissociation of negative regulators of the complex.

scholarly journals Chaperonins

1998 ◽  
Vol 333 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Neil A. RANSON ◽  
Helen E. WHITE ◽  
Helen R. SAIBIL
Keyword(s):  
Conformational Changes ◽  
Substrate Binding ◽  
Atp Binding ◽  
Dependent Manner ◽  
Oligomeric Proteins ◽  
Double Ring ◽  
Substrate Protein ◽  
Hydrophobic Binding ◽  
Substrate Binding Sites

The molecular chaperones are a diverse set of protein families required for the correct folding, transport and degradation of other proteins in vivo. There has been great progress in understanding the structure and mechanism of action of the chaperonin family, exemplified by Escherichia coli GroEL. The chaperonins are large, double-ring oligomeric proteins that act as containers for the folding of other protein subunits. Together with its co-protein GroES, GroEL binds non-native polypeptides and facilitates their refolding in an ATP-dependent manner. The action of the ATPase cycle causes the substrate-binding surface of GroEL to alternate in character between hydrophobic (binding/unfolding) and hydrophilic (release/folding). ATP binding initiates a series of dramatic conformational changes that bury the substrate-binding sites, lowering the affinity for non-native polypeptide. In the presence of ATP, GroES binds to GroEL, forming a large chamber that encapsulates substrate proteins for folding. For proteins whose folding is absolutely dependent on the full GroE system, ATP binding (but not hydrolysis) in the encapsulating ring is needed to initiate protein folding. Similarly, ATP binding, but not hydrolysis, in the opposite GroEL ring is needed to release GroES, thus opening the chamber. If the released substrate protein is still not correctly folded, it will go through another round of interaction with GroEL.

scholarly journals Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line

1987 ◽  
Vol 247 (3) ◽  
pp. 731-738 ◽  
Author(s):  
S L Swope ◽  
A Schonbrunn
Keyword(s):  
Islet Cells ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Single Class ◽  
Relative Potencies ◽  
Stimulate Insulin Release ◽  
Bombesin Receptors

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The m6A “reader” YTHDF1 promotes osteogenesis of bone marrow mesenchymal stem cells through translational control of ZNF839

2021 ◽  
Vol 12 (11) ◽  
Author(s):  
Tao Liu ◽  
Xinfeng Zheng ◽  
Chenglong Wang ◽  
Chuandong Wang ◽  
Shengdan Jiang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Translational Control ◽  
Zinc Finger Protein ◽  
Dependent Manner ◽  
Marrow Mesenchymal Stem Cells ◽  
Rna Immunoprecipitation

AbstractN6-methyladenosine (m6A) is required for differentiation of human bone marrow mesenchymal stem cells (hBMSCs). However, its intrinsic mechanisms are largely unknown. To identify the possible role of m6A binding protein YTHDF1 in hBMSCs osteogenesis in vivo, we constructed Ythdf1 KO mice and showed that depletion of Ythdf1 would result in decreased bone mass in vivo. Both deletion of Ythdf1 in mouse BMSCs and shRNA-mediated knockdown of YTHDF1 in hBMSCs prevented osteogenic differentiation of cells in vitro. Using methylated RNA immunoprecipitation (Me-RIP) sequencing and RIP-sequencing, we found that ZNF839 (a zinc finger protein) served as a target of YTHDF1. We also verified its mouse homolog, Zfp839, was translationally regulated by Ythdf1 in an m6A-dependent manner. Zfp839 potentiated BMSC osteogenesis by interacting with and further enhancing the transcription activity of Runx2. These findings should improve our understanding of the mechanism of BMSC osteogenesis regulation and provide new ideas for the prevention and treatment of osteoporosis.

scholarly journals Properties of the Naturally Occurring Soluble Surface Glycoprotein of Ecotropic Murine Leukemia Virus: Binding Specificity and Possible Conformational Change after Binding to Receptor

2000 ◽  
Vol 74 (4) ◽  
pp. 1815-1826 ◽  
Author(s):  
Hidetoshi Ikeda ◽  
Kanako Kato ◽  
Takako Suzuki ◽  
Hiroshi Kitani ◽  
Yutaka Matsubara ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Surface Glycoprotein ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Naturally Occurring ◽  
Mouse Cells ◽  
Murine Leukemia

ABSTRACT Ecotropic murine leukemia virus (MuLV) infection is initiated by the interaction between the surface glycoprotein (SU) of the virus and its cell-surface receptor mCAT-1. We investigated the SU-receptor interaction by using a naturally occurring soluble SU which was encoded by the envelope (env) gene of a defective endogenous MuLV, Fv-4r . Binding of the SU to mCAT-1-positive mouse cells was completed by 1 min at 37°C. The SU could not bind to mouse cells that were persistently infected by ecotropic MuLVs (but not amphotropic or dualtropic MuLVs) or transfected with wild-type ecotropic env genes or a mutantenv gene which can express only precursor Env protein that is restricted to retention in the endoplasmic reticulum. These cells were also resistant to superinfection by ecotropic MuLVs. Thus, superinfection resistance correlated with the lack of SU-binding capacity. After binding to the cells, the SU appeared to undergo some conformational changes within 1 min in a temperature-dependent manner. This was suggested by the different properties of two monoclonal antibodies (MAbs) reactive with the same C-terminal half of theFv-4r SU domain, including a proline-rich motif which was shown to be important for conformation of the SU and interaction between the SU and the transmembrane protein. One MAb reacting with the soluble SU bound to cells was dissociated by a temperature shift from 4 to 37°C. Such dissociation was not observed in cells synthesizing the SU or when another MAb was used, indicating that the dissociation was not due to a temperature-dependent release of the MAb but to possible conformational changes in the SU.

scholarly journals Iron in cytosolic ferritin can be recycled through lysosomal degradation in human fibroblasts

1998 ◽  
Vol 336 (1) ◽  
pp. 201-205 ◽  
Author(s):  
Derek C. RADISKY ◽  
Jerry KAPLAN
Keyword(s):  
Human Fibroblasts ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
Temperature Dependent ◽  
Intracellular Iron ◽  
Vitro Assay ◽  
Cellular Iron ◽  
Horse Spleen Ferritin

Examination of the mechanism of intracellular iron recovery from lysosomally-degraded ferritin in vivo has been complicated by the continuous flux of cellular iron through ferritin molecules. Here we incubated human fibroblasts with cationic ferritin, a derivative of horse spleen ferritin, as a technique for delivering immunologically distinct ferritin molecules directly to lysosomes. Using this method, we found increased endogenous ferritin levels after the cellular degradation of cationic ferritin, demonstrating that cells can utilize lysosomal ferritin to produce increased cytosolic ferritin levels. Further, using an in vitro assay, we showed that isolated lysosomes degrade endogenous ferritin in a time- and temperature-dependent manner. These results are consistent with a model in which cytosolic ferritin is taken into the lysosomes and degraded. The solubilized iron from the ferric core could then be transported across the lysosomal membrane back into the cytosol.

scholarly journals mRNA- and Factor-Driven Dynamic Variability Controls eIF4F-Cap Recognition for Translation Initiation.

2021 ◽  
Author(s):  
Burak Çetin ◽  
Seán E. O'Leary
Keyword(s):  
Single Molecule ◽  
Translational Control ◽  
Atp Hydrolysis ◽  
Translation Efficiency ◽  
Dependent Manner ◽  
Complex Interplay ◽  
Eif4f Complex ◽  
Mrna Length ◽  
Mrna Binding

mRNA 5′ cap recognition by eIF4F is a key step in eukaryotic translational control. While different mRNAs respond differently to eIF4F–directed regulation, the molecular basis for this variability remains unclear. We developed single-molecule fluorescence assays to directly observe eIF4F–mRNA interactions. We uncovered a complex interplay of mRNA features with factor activities that differentiates cap recognition between mRNAs. eIF4E–cap association rates are anticorrelated with mRNA length. eIF4A leverages ATP binding to differentially accelerate eIF4E–mRNA association; the extent of this acceleration correlates with translation efficiency in vivo. eIF4G lengthens eIF4E–cap binding to persist on the initiation timescale. The full eIF4F complex discriminates between mRNAs in an ATP-dependent manner. After eIF4F–mRNA binding, eIF4E is ejected from the cap by eIF4A ATP hydrolysis. Our results suggest features throughout mRNA coordinate in controlling cap recognition at the 5ʹ end, and suggest a model for how eIF4F–mRNA dynamics establish mRNA sensitivity to translational control processes.

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