Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Translation dynamics of single mRNAs in live cells and neurons.

ABSTRACT: Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single-molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display "bursting" translation. This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells.

SUBMITTER: Wu B

PROVIDER: S-EPMC4939616 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Translation dynamics of single mRNAs in live cells and neurons.

Wu Bin B Eliscovich Carolina C Yoon Young J YJ Singer Robert H RH

Science (New York, N.Y.) 20160505 6292

Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are ...[more]

PMID: 27313041

Similar Datasets

Bursting translation on single mRNAs in live cells.

Project description:Stochasticity has emerged as a mechanism of gene regulation. Much of this so-called "noise" has been attributed to bursting transcription. Although bursting transcription has been studied extensively, the role of stochasticity in translation has not been fully investigated due to the lack of enabling imaging technology. In this study, we developed techniques to track single mRNAs and their translation in live cells for hours, allowing the measurement of previously uncharacterized translation dynamics. We applied genetic and pharmacological perturbations to control translation kinetics and found that, like transcription, translation is not a constitutive process but instead cycles between inactive and active states, or "bursts." However, unlike transcription, which is largely frequency-modulated, complex structures in the 5'-untranslated region alter burst amplitudes. Bursting frequency can be controlled through cap-proximal sequences and trans-acting factors such as eIF4F. We coupled single-molecule imaging with stochastic modeling to quantitatively determine the kinetic parameters of translational bursting.

| S-EPMC10330622 | biostudies-literature

Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells.

Project description:Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13-18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation. Remarkably, tagging of the heavy chain of dynein 1 (DYNC1H1) shows this mRNA accumulates in foci containing three to seven RNA molecules. These foci are translation sites and thus represent specialized translation factories. We also observe that DYNC1H1 polysomes are actively transported by motors, which may deliver the mature protein at appropriate cellular locations. The SunTag should be broadly applicable to study translational regulation in live single cells.

| S-EPMC5021097 | biostudies-literature

Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells.

Project description:Viruses use internal ribosome entry sites (IRES) to hijack host ribosomes and promote cap-independent translation. Although they are well-studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5' cap, and the other from the encephalomyocarditis virus IRES. When combined with a pair of complementary probes that bind the epitopes cotranslationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap-dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively, our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states.

| S-EPMC8351040 | biostudies-literature

Spatiotemporal control of translation in live zebrafish embryos via photoprotected mRNAs.

Project description:Translation of mRNA into protein is a fundamental process and tightly controlled during development. Several mechanisms acting on the mRNA level regulate when and where an mRNA is expressed. To explore the effects of conditional and transient gene expression in a developing organism, it is vital to experimentally enable abrogation and restoration of translation. We recently developed the FlashCaps technology allowing preparation of translationally muted mRNAs and their controlled activation by light. Here, we validate its functionality in vivo. We demonstrate that translation of FlashCap-eGFP-mRNA can be triggered in zebrafish embryos with spatiotemporal control. The injected FlashCap-mRNA is stable for hours and remains muted. Light-mediated activation up to 24 h post fertilization produces visible amounts of eGFP and can be restricted to distinct parts of the embryo. This methodology extends the toolbox for vertebrate models by enabling researchers to locally activate mRNA translation at different timepoints during development.

| S-EPMC11743775 | biostudies-literature

Background free imaging of single mRNAs in live cells using split fluorescent proteins.

Project description:We describe a technique for imaging single mRNAs in living cells based on fluorescent protein (FP) complementation. We employ the high affinity interaction between the bacterial phage MS2/PP7 coat proteins and their respective RNA binding motifs as an RNA scaffold to bring two halves of a split-FP together to image single reporter mRNAs without background fluorescence.

| S-EPMC3885884 | biostudies-literature

Real-Time Imaging of Translation on Single mRNA Transcripts in Live Cells.

Project description:Translation is under tight spatial and temporal controls to ensure protein production in the right time and place in cells. Methods that allow real-time, high-resolution visualization of translation in live cells are essential for understanding the spatiotemporal dynamics of translation regulation. Based on multivalent fluorescence amplification of the nascent polypeptide signal, we develop a method to image translation on individual mRNA molecules in real time in live cells, allowing direct visualization of translation events at the translation sites. Using this approach, we monitor transient changes of translation dynamics in responses to environmental stresses, capture distinct mobilities of individual polysomes in different subcellular compartments, and detect 3' UTR-dependent local translation and active transport of polysomes in dendrites of primary neurons.

| S-EPMC4905760 | biostudies-literature

Single-molecule imaging reveals translation-dependent destabilization of mRNAs.

Project description:The relationship between mRNA translation and decay is incompletely understood, with conflicting reports suggesting that translation can either promote decay or stabilize mRNAs. The effect of translation on mRNA decay has mainly been studied using ensemble measurements and global transcription and translation inhibitors, which can have pleiotropic effects. We developed a single-molecule imaging approach to control the translation of a specific transcript that enabled simultaneous measurement of translation and mRNA decay. Our results demonstrate that mRNA translation reduces mRNA stability, and mathematical modeling suggests that this process is dependent on ribosome flux. Furthermore, our results indicate that miRNAs mediate efficient degradation of both translating and non-translating target mRNAs and reveal a predominant role for mRNA degradation in miRNA-mediated regulation. Simultaneous observation of translation and decay of single mRNAs provides a framework to directly study how these processes are interconnected in cells.

| S-EPMC9957601 | biostudies-literature

A live-imaging protocol for tracking receptor dynamics in single cells.

Project description:Adjacent membrane receptors can show different cellular responses to ligand stimulation. Here, we describe a super-resolution microscopy imaging protocol for tracking the dynamics of two different membrane-bound receptors in single cells. We describe the transfection protocol by electroporation. We detail the imaging procedure for receptors in a single cell. We then outline the data analysis pipeline. We have applied this protocol to imaging of endocytosis of the LOX-1 and AT1 in CHO-K1 cells, but the protocol can be applied to a variety of membrane receptors in other cell lines. For complete details on the use and execution of this protocol, please refer to Takahashi et al. (2021).

| S-EPMC9059088 | biostudies-literature

Fluorophore-Promoted RNA Folding and Photostability Enables Imaging of Single Broccoli-Tagged mRNAs in Live Mammalian Cells.

Project description:Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli-DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli-BI complexes in cells. Broccoli-BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis-BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.

| S-EPMC8158785 | biostudies-literature

Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells.

Project description:PolC is one of two essential replicative DNA polymerases found in the Gram-positive bacterium Bacillus subtilis. The B. subtilis replisome is eukaryotic-like in that it relies on a two DNA polymerase system for chromosomal replication. To quantitatively image how the replicative DNA polymerase PolC functions in B. subtilis, we applied photobleaching-assisted microscopy, three-dimensional superresolution imaging, and single-particle tracking to examine the in vivo behavior of PolC at single-molecule resolution. We report the stoichiometry of PolC proteins within each cell and within each replisome, we elucidate the diffusion characteristics of individual PolC molecules, and we quantify the exchange dynamics for PolC engaged in lagging strand synthesis. We show that PolC is highly dynamic: this DNA polymerase is constantly recruited to and released from a centrally located replisome, providing, to our knowledge, new insight into the organization and dynamics of the replisome in bacterial cells.

| S-EPMC5192695 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data