scholarly journals Characterizing the structure-function relationship of a naturally-occurring RNA thermometer

2017 ◽  
Author(s):  
Sarai Meyer ◽  
Julius B. Lucks
Keyword(s):  
Heat Shock ◽  
Rna Structure ◽  
Structural Changes ◽  
Structural Motif ◽  
Ribosome Binding ◽  
Heat Shock Responses ◽  
Function Relationship ◽  
Relationship Of

AbstractA wide number of bacteria have been found to govern virulence and heat shock responses using temperature-sensing RNAs known as RNA thermometers. A prime example is theagsAthermometer known to regulate the production of the AgsA heat shock protein inSalmonella entericausing a “fourU” structural motif. Using the SHAPE-Seq RNA structure-probing methodin vivoandin vitro, we found that the regulator functions by a subtle shift in equilibrium RNA structure populations that lead to a partial melting of the helix containing the ribosome binding site. We also demonstrate that ribosome binding to theagsAmRNA causes changes to the thermometer structure that appear to facilitate thermometer helix unwinding. These results demonstrate how subtle RNA structural changes can govern gene expression and illuminate the function of an important bacterial regulatory motif.

scholarly journals New multiscale characterisation methodology for effective determination of isolation-structure-function relationship of extracellular vesicles

2021 ◽  
Author(s):  
Thanh Huyen Phan ◽  
Shiva Kamini Divakarla ◽  
Jia Hao Yeo ◽  
Qingyu Lei ◽  
Priyanka Tharkar ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biological Effects ◽  
Dry Mass ◽  
Experimental Methodology ◽  
Wide Range ◽  
Isolation Methods ◽  
Function Relationship ◽  
Relationship Of

AbstractExtracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass-produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterisation approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

scholarly journals Genome-wide RNA structurome reprogramming by acute heat shock globally regulates mRNA abundance

2018 ◽  
Vol 115 (48) ◽  
pp. 12170-12175 ◽  
Author(s):  
Zhao Su ◽  
Yin Tang ◽  
Laura E. Ritchey ◽  
David C. Tack ◽  
Mengmeng Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Rna Structure ◽  
Time Course ◽  
Structural Changes ◽  
Dimethyl Sulfate ◽  
Rna Degradation ◽  
Crop Species ◽  
Genome Wide

The heat shock response is crucial for organism survival in natural environments. RNA structure is known to influence numerous processes related to gene expression, but there have been few studies on the global RNA structurome as it prevails in vivo. Moreover, how heat shock rapidly affects RNA structure genome-wide in living systems remains unknown. We report here in vivo heat-regulated RNA structuromes. We applied Structure-seq chemical [dimethyl sulfate (DMS)] structure probing to rice (Oryza sativa L.) seedlings with and without 10 min of 42 °C heat shock and obtained structural data on >14,000 mRNAs. We show that RNA secondary structure broadly regulates gene expression in response to heat shock in this essential crop species. Our results indicate significant heat-induced elevation of DMS reactivity in the global transcriptome, revealing RNA unfolding over this biological temperature range. Our parallel Ribo-seq analysis provides no evidence for a correlation between RNA unfolding and heat-induced changes in translation, in contrast to the paradigm established in prokaryotes, wherein melting of RNA thermometers promotes translation. Instead, we find that heat-induced DMS reactivity increases correlate with significant decreases in transcript abundance, as quantified from an RNA-seq time course, indicating that mRNA unfolding promotes transcript degradation. The mechanistic basis for this outcome appears to be mRNA unfolding at both 5′ and 3′-UTRs that facilitates access to the RNA degradation machinery. Our results thus reveal unexpected paradigms governing RNA structural changes and the eukaryotic RNA life cycle.

scholarly journals Insights on the Quest for the Structure–Function Relationship of the Mitochondrial Pyruvate Carrier

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 407
Author(s):  
José Edwin Neciosup Quesñay ◽  
Naomi L. Pollock ◽  
Raghavendra Sashi Krishna Nagampalli ◽  
Sarah C. Lee ◽  
Vijayakumar Balakrishnan ◽  
...  
Keyword(s):  
Molecular Structures ◽  
Computational Tools ◽  
Protein Subunits ◽  
Molecular Identity ◽  
Current State ◽  
Mitochondrial Pyruvate Carrier ◽  
Function Relationship ◽  
Relationship Of

The molecular identity of the mitochondrial pyruvate carrier (MPC) was presented in 2012, forty years after the active transport of cytosolic pyruvate into the mitochondrial matrix was first demonstrated. An impressive amount of in vivo and in vitro studies has since revealed an unexpected interplay between one, two, or even three protein subunits defining different functional MPC assemblies in a metabolic-specific context. These have clear implications in cell homeostasis and disease, and on the development of future therapies. Despite intensive efforts by different research groups using state-of-the-art computational tools and experimental techniques, MPCs’ structure-based mechanism remains elusive. Here, we review the current state of knowledge concerning MPCs’ molecular structures by examining both earlier and recent studies and presenting novel data to identify the regulatory, structural, and core transport activities to each of the known MPC subunits. We also discuss the potential application of cryogenic electron microscopy (cryo-EM) studies of MPC reconstituted into nanodiscs of synthetic copolymers for solving human MPC2.

scholarly journals Modular Structure of PACT: Distinct Domains for Binding and Activating PKR

2001 ◽  
Vol 21 (6) ◽  
pp. 1908-1920 ◽  
Author(s):  
Gregory A. Peters ◽  
Rune Hartmann ◽  
Jun Qin ◽  
Ganes C. Sen
Keyword(s):  
Protein Domain ◽  
Affinity Binding ◽  
Translation Inhibition ◽  
High Affinity ◽  
Cellular Apoptosis ◽  
Domain 3 ◽  
Function Relationship ◽  
Relationship Of

ABSTRACT PACT is a 35-kDa human protein that can directly bind and activate the latent protein kinase, PKR. Here we report that PKR activation by PACT causes cellular apoptosis in addition to PKR autophosphorylation and translation inhibition. We analyzed the structure-function relationship of PACT by measuring its ability to bind and activate PKR in vitro and in vivo. Our studies revealed that among three domains of PACT, the presence of either domain 1 or domain 2 was sufficient for high-affinity binding of PACT to PKR. On the other hand, domain 3, consisting of 66 residues, was absolutely required for PKR activation in vitro and in vivo. When fused to maltose-binding protein, domain 3 was also sufficient for efficiently activating PKR in vitro. However, it bound poorly to PKR at the physiological salt concentration and consequently could not activate it properly in vivo. As anticipated, activation of PKR by domain 3 in vivo could be restored by attaching it to a heterologous PKR-binding domain. These results demonstrated that the structure of PACT is modular: it is composed of a distinct PKR-activation domain and two mutually redundant PKR-interacting domains.

Alterations in the pattern of gene expression following heat shock in the nematode Caenorhabditis elegans

1983 ◽  
Vol 61 (6) ◽  
pp. 480-487 ◽  
Author(s):  
Terry P. Snutch ◽  
David L. Baillie
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Elevated Temperatures ◽  
In Vitro Study ◽  
In Vitro Translation ◽  
Nematode Caenorhabditis Elegans ◽  
Stage Of Development ◽  
Heat Shock Responses

Exposure of the nematode Caenorhabditis elegans to elevated temperatures induces the preferential synthesis of eight major polypeptides of approximate molecular weights 81 000, 70 000, 41 000, 38 000, 29 000, 19 000, 18 000, and 16 000. In pulse-labelled worms these peptides first appear at 29 °C and continue to be synthesized up to lethal temperatures. They are heat inducible at every stage of development. While temperature elevation induces the synthesis of the heat-shock polypeptides, the in vivo synthesis of most other proteins present before heat shock is suppressed. In contrast, in vitro translation of mRNA from heat-shocked worms shows no alteration from the pattern of normal 20 °C mRNAs except for the appearance of the heat-shock mRNAs. An in vitro study of RNA from control and heat-shocked dauer larvae shows that this developmental variant possesses little translatable mRNA but, upon heat shock, synthesizes a set of messages corresponding to the heat-shock polypeptides. The low background of this system will be especially useful in the analysis and purification of heat-shock mRNA for molecular cloning experiments. Extensive similarities between the Drosophila and C. elegans heat-shock responses are shown, including homology between the 70-kdalton heat-shock genes of the two organisms.

scholarly journals New Multiscale Characterization Methodology for Effective Determination of Isolation–Structure–Function Relationship of Extracellular Vesicles

2021 ◽  
Vol 9 ◽  
Author(s):  
Thanh Huyen Phan ◽  
Shiva Kamini Divakarla ◽  
Jia Hao Yeo ◽  
Qingyu Lei ◽  
Priyanka Tharkar ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biological Effects ◽  
Dry Mass ◽  
Experimental Methodology ◽  
Wide Range ◽  
Isolation Methods ◽  
Function Relationship ◽  
Relationship Of

Extracellular vesicles (EVs) have been lauded as next-generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass produce EVs, i.e., to isolate, purify, and characterize them effectively. Technical limitations in comprehensive characterization of EVs lead to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were likely to contribute to the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterization approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

scholarly journals The structure-function relationship of oncogenic LMTK3

2020 ◽  
Vol 6 (46) ◽  
pp. eabc3099
Author(s):  
Angeliki Ditsiou ◽  
Chiara Cilibrasi ◽  
Nikiana Simigdala ◽  
Athanasios Papakyriakou ◽  
Leanne Milton-Harris ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Heat Shock Protein 90 ◽  
Kinase Domain ◽  
Time Resolved ◽  
Molecule Inhibitor ◽  
Effective Doses ◽  
Function Relationship ◽  
Relationship Of

Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.

Sign in / Sign up

Export Citation Format

Share Document