scholarly journals Global analysis of gene expression dynamics identifies factors required for accelerated mRNA degradation

2018 ◽  
Author(s):  
Darach Miller ◽  
Nathan Brandt ◽  
David Gresham
Keyword(s):  
Single Molecule ◽  
Translational Control ◽  
Environmental Changes ◽  
Global Analysis ◽  
Mrna Degradation ◽  
Branched Dna ◽  
Degradation Rates ◽  
Gene Expression Dynamics ◽  
Mrna Destabilization ◽  
Barcode Sequencing

AbstractCellular responses to changing environments frequently involve rapid reprogramming of the transcriptome. Regulated changes in mRNA degradation rates can accelerate reprogramming by clearing or stabilizing extant transcripts. Here, we measured mRNA stability using 4-thiouracil labeling in the budding yeastSaccharomyces cerevisiaeduring a nitrogen upshift and found that 78 mRNAs are subject to destabilization. These transcripts include Nitrogen Catabolite Repression (NCR) and carbon metabolism mRNAs, suggesting that mRNA destabilization is a mechanism for targeted reprogramming. To explore the molecular basis of destabilization we implemented a SortSeq approach to screen using the pooled deletion collection library fortransfactors that mediate rapidGAP1mRNA repression. We combined low-input multiplexed Barcode sequencing with branched-DNA single-molecule mRNA FISH and Fluorescence-activated cell sorting (BFF) to identify that the Lsm1-7p/Pat1p complex and general mRNA decay machinery are important forGAP1mRNA clearance. We also find that the decapping modulatorSCD6,translation factor eIF4G2, and the 5’ UTR ofGAP1are important for this repression, suggesting that translational control may impact the post-transcriptional fate of mRNAs in response to environmental changes.

scholarly journals Use of Citizen Science Data for Characterizing Patterns and Drivers of Avian Altitudinal Migration

2019 ◽  
Vol 3 ◽  
Author(s):  
Pei-Yu Tsai ◽  
Chie-Jen Ko ◽  
Ya-Jung Lu ◽  
Chia Hsieh ◽  
Mao-Ning Tuanmu
Keyword(s):  
Citizen Science ◽  
Environmental Changes ◽  
Global Analysis ◽  
Research Question ◽  
Bird Species ◽  
Migration Behavior ◽  
Mountain Regions ◽  
Science Data ◽  
Essential Information ◽  
Altitudinal Migration

Altitudinal migration, the seasonal and repeateing movement of animal individuals between breeding and non-breeding areas at different elevations, is a common and important but understudied behavior in birds. Difficulty in characterizing avian altitudinal migration has prevented a comprehensive understanding of both patterns and drivers of this behavior. To fill this knowledge gap, we investigated altitudinal migration patterns and underlying mechanisms for a major proportion (~70%) of an entire resident bird community on a subtropical island with an almost 4000-m elevational gradient. We quantified migration tendency of individual bird species based on the seasonal shift in the elevational distribution of their occurrence records in the eBird database. We then built phylogeny-controlled regression models to examine the associations between the birds’ migration tendencies and their functional traits to test major hypotheses on the mechanisms of altitudinal migration. The results showed a common but variable altitudinal migration behavior among the 118 species examined, with 40 and 11 species conducting post-breeding downhill and uphill migration, respectively. The species that have a narrower thermal tolerance range, can tolerate lower temperatures, have a smaller body size, have a more diverse or invertebrate-rich diet, or use an open nest had a higher downhill migration tendency. In contrast, no traits examined showed consistent associations with the uphill migration tendency. This suggests that post-breeding downhill and uphill migrations are driven by different processes and current hypotheses can only explain the former, but not the latter. This relatively comprehensive study demonstrated the power of citizen science data to provide new insights into an old research question from a novel perspective. Using the same approach, we are investigating the behavior in mountain regions around the world. With the global analysis, we will be able to understand the general patterns and mechanisms of avian altitudinal migration and also investigate their variation among mountain regions in different climate zones. In the face of rapid environmental changes in mountain ecosystems, the approach used in this study may also provide essential information for the conservation of mountainous biodiversity.

scholarly journals mRNA structural dynamics shape Argonaute-target interactions

2019 ◽  
Author(s):  
Suzan Ruijtenberg ◽  
Stijn Sonneveld ◽  
Tao Ju Cui ◽  
Ive Logister ◽  
Dion de Steenwinkel ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
Protein Interactions ◽  
Small Rnas ◽  
Mrna Degradation ◽  
Translational Repression ◽  
Modeling And Experiments ◽  
Regulate Protein ◽  
Sirna Target

AbstractSmall RNAs (such as miRNAs, siRNAs and piRNAs) regulate protein expression in a wide variety of biological processes and play an important role in cellular function, development and disease. Association of small RNAs with Argonaute (AGO) family proteins guide AGO to target RNAs, generally resulting in target silencing through transcriptional silencing, translational repression or mRNA degradation. Here we develop a live-cell single-molecule imaging assay to simultaneously visualize translation of individual mRNA molecules and their silencing by human AGO2-siRNA complexes. We find that siRNA target sites are commonly masked in vivo by RNA secondary structures, which inhibit AGO2-target interactions. Translating ribosomes unmask AGO2 binding sites, stimulating AGO2-target interactions and promoting mRNA degradation. Using a combination of mathematical modeling and experiments, we find that mRNA structures are highly heterogeneous and continuously refolding. We show that structural dynamics of mRNAs shape AGO2-target recognition, which may be a common feature controlling mRNA-protein interactions.

scholarly journals Real-Time 3D Imaging at the Single Molecule Level of Signal Transduction in Saccharomyces CerevisiÆ Responding to Environmental Changes

2016 ◽  
Vol 110 (3) ◽  
pp. 145a
Author(s):  
Erik G. Hedlund ◽  
Sviatlana Shashkova ◽  
Adam J.M. Wollman ◽  
Stefan Hohmann ◽  
Mark C. Leake
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Transduction ◽  
Real Time ◽  
Single Molecule ◽  
3D Imaging ◽  
Environmental Changes ◽  
Single Molecule Level

scholarly journals Initiation factor 2 stabilizes the ribosome in a semirotated conformation

2015 ◽  
Vol 112 (52) ◽  
pp. 15874-15879 ◽  
Author(s):  
Clarence Ling ◽  
Dmitri N. Ermolenko
Keyword(s):  
Single Molecule ◽  
Translational Control ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Bound State ◽  
Initiation Factor ◽  
Large Ribosomal Subunit ◽  
Control Of Gene Expression ◽  
Initiation Phase ◽  
Intersubunit Rotation

Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNAfMet, and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding.

scholarly journals Global Analysis of the Zinc Homeostasis Network in Pseudomonas aeruginosa and Its Gene Expression Dynamics

2021 ◽  
Vol 12 ◽  
Author(s):  
Verena Ducret ◽  
Melina Abdou ◽  
Catarina Goncalves Milho ◽  
Sara Leoni ◽  
Oriane Martin--Pelaud ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Global Analysis ◽  
Opportunistic Pathogen ◽  
Zinc Homeostasis ◽  
Protein Levels ◽  
Its Gene ◽  
Zn Homeostasis ◽  
Gene Expression Dynamics ◽  
Nanostring Technology

Zinc is one of the most important trace elements for life and its deficiency, like its excess, can be fatal. In the bacterial opportunistic pathogen Pseudomonas aeruginosa, Zn homeostasis is not only required for survival, but also for virulence and antibiotic resistance. Thus, the bacterium possesses multiple Zn import/export/storage systems. In this work, we determine the expression dynamics of the entire P. aeruginosa Zn homeostasis network at both transcript and protein levels. Precisely, we followed the switch from a Zn-deficient environment, mimicking the initial immune strategy to counteract bacterial infections, to a Zn-rich environment, representing the phagocyte metal boost used to eliminate an engulfed pathogen. Thanks to the use of the NanoString technology, we timed the global silencing of Zn import systems and the orchestrated induction of Zn export systems. We show that the induction of Zn export systems is hierarchically organized as a function of their impact on Zn homeostasis. Moreover, we identify PA2807 as a novel Zn resistance component in P. aeruginosa and highlight new regulatory links among Zn-homeostasis systems. Altogether, this work unveils a sophisticated and adaptive homeostasis network, which complexity is key in determining a pathogen spread in the environment and during host-colonization.

scholarly journals Single-molecule imaging of mRNA localization and regulation during the integrated stress response

2018 ◽  
Author(s):  
Johannes H. Wilbertz ◽  
Franka Voigt ◽  
Ivana Horvathova ◽  
Gregory Roth ◽  
Yinxiu Zhan ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Molecule ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Localization ◽  
Mrna Degradation ◽  
Integrated Stress Response ◽  
Cellular Space ◽  
Processing Bodies ◽  
Functional Consequence

AbstractBiological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules(SGs) and processing bodies(PBs), which are distinct biomolecular condensates, and its effect on translation and mRNA degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics and that specific RNA binding proteins can anchor mRNAs within these compartments. During recovery from stress, mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts.

scholarly journals Metabolic labelling of RNA uncovers the contribution of transcription and decay rates on hypoxia-induced changes in RNA levels

2019 ◽  
Author(s):  
Maria Tiana ◽  
Clara Galiana ◽  
Miguel Ángel Fernández-Moreno ◽  
Benilde Jimenez ◽  
Luis del Peso
Keyword(s):  
Rna Synthesis ◽  
Environmental Changes ◽  
Hypoxia Inducible Factor ◽  
Rna Degradation ◽  
Decay Rates ◽  
Specific Gene ◽  
Metabolic Labeling ◽  
Low Oxygen ◽  
Degradation Rates ◽  
Rna Levels

ABSTRACTCells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension. We found that the changes in transcription rates induced by hypoxia are the major determinant of RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half-life in normoxia of 8.7 hours, to 5.7 hours in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect was mimicked by forced activation of the Hypoxia Inducible Factor pathway and prevented by its interference. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter.

scholarly journals Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin

2021 ◽  
Author(s):  
Lina Hamouche ◽  
Leonora Poljak ◽  
Agamemnon J. Carpousis
Keyword(s):  
16S Rrna ◽  
Rna Polymerase ◽  
Growth Conditions ◽  
Rna Degradation ◽  
Mrna Degradation ◽  
Rnase E ◽  
Rifampicin Treatment ◽  
Bacterial Rna ◽  
Degradation Rates ◽  
Rrna Degradation

AbstractRifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete thus permitting the re-initiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.

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