Measuring mRNA translation in neuronal processes and somata by tRNA-FRET

Abstract In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

Download Full-text

Measuring mRNA translation in neuronal processes and somata by tRNA-FRET

10.1101/646216 ◽

2019 ◽

Author(s):

Bella Koltun ◽

Sivan Ironi ◽

Noga Gershoni-Emek ◽

Iliana Barrera ◽

Mohammad Hleihil ◽

...

Keyword(s):

Protein Synthesis ◽

Mrna Translation ◽

Chemical Induction ◽

Translational Machinery ◽

Disease States ◽

Neural Processes ◽

Neuronal Processes ◽

Health And Disease ◽

Slow Transport ◽

Temporal Localization

AbstractIn neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. In this work, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and movement of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA motion in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of LTP in culture revealed GluR-dependent biphasic up-regulation of mRNA translation with a similar effect in dendrites and somata. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

Download Full-text

Astrocytes locally translate transcripts in their peripheral processes

10.1101/071795 ◽

2016 ◽

Author(s):

Kristina Sakers ◽

Allison M. Lake ◽

Rohan Khazanchi ◽

Rebecca Ouwenga ◽

Michael J. Vasek ◽

...

Keyword(s):

De Novo ◽

Long Term Potentiation ◽

Acid Synthesis ◽

Local Translation ◽

Sequence Dependent ◽

Specific Subset ◽

Term Potentiation ◽

Neuronal Processes ◽

Cellular Compartments ◽

Computational Analyses

AbstractLocal translation in neuronal processes is key to the alteration of synaptic strength that contributes to long term potentiation and learning and memory. Here, we present evidence that astrocytes have ribosomes in their peripheral and perisynaptic processes, and that de novo protein synthesis occurs in the astrocyte periphery. We also developed a new biochemical approach to profile and define a set of candidate transcripts that are locally translated in astrocyte processes, several of which were validated in vivo using in situ hybridization of sparsely labeled cells. Computational analyses indicate that localized translation is both sequence dependent and enriched for particular biological functions. This includes novel pathways such as fatty acid synthesis as well as pathways consistent with known roles for astrocyte processes, such as GABA and glutamate metabolism. Finally, enriched transcripts also include key glial regulators of synaptic refinement, suggesting that local production of astrocyte proteins may support microscale alterations of adjacent synapses.Significance StatementCellular compartments are specialized for particular functions. In astrocytes, the peripheral processes, particularly near synapses, contain proteins specialized for reuptake of neurotransmitters and ions, and have been shown to alter their morphology in response to activity. Regulated transport of a specific subset of nuclear-derived mRNAs to particular compartments is thought to support the specialization of these compartments and allow for local regulation of translation. In neurons, local translation near activated synapses is thought to generate the proteins needed for the synaptic alterations that constitute memory. We demonstrate that astrocytes also have sequence-dependent local translation in their peripheral processes, including transcripts with roles in regulating synapses. This suggests local translation in astrocyte processes may also play a role in modulating synapses.

Download Full-text

A Multi-Phase Model of Synaptic Long-Term Potentiation in Hippocampal CA1 Neurones: Protein Kinase C Activation and Protein Synthesis Are Required for the Maintenance of the Trace

Synaptic Plasticity in the Hippocampus ◽

10.1007/978-3-642-73202-7_37 ◽

1988 ◽

pp. 126-129 ◽

Cited By ~ 3

Author(s):

K. Reymann ◽

U. Frey ◽

H. Matthies

Keyword(s):

Protein Synthesis ◽

Protein Kinase C ◽

Protein Kinase ◽

Long Term Potentiation ◽

Kinase C ◽

Term Potentiation ◽

Hippocampal Ca1 ◽

Multi Phase ◽

Protein Kinase C Activation

Download Full-text

Selective targeting of mRNA and the following protein synthesis of CaMKIIα at the long-term potentiation-induced site

Biology Open ◽

10.1242/bio.042861 ◽

2019 ◽

Vol 9 (1) ◽

pp. bio042861

Author(s):

Itsuko Nihonmatsu ◽

Noriaki Ohkawa ◽

Yoshito Saitoh ◽

Reiko Okubo-Suzuki ◽

Kaoru Inokuchi

Keyword(s):

Protein Synthesis ◽

Long Term Potentiation ◽

Term Potentiation ◽

Selective Targeting

Download Full-text

Cytoplasmic Polyadenylation in Development and Beyond

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.63.2.446-456.1999 ◽

1999 ◽

Vol 63 (2) ◽

pp. 446-456 ◽

Cited By ~ 176

Author(s):

Joel D. Richter

Keyword(s):

Mrna Translation ◽

Long Term Potentiation ◽

Evolutionary Conservation ◽

Adult Brain ◽

Cis Elements ◽

Cytoplasmic Polyadenylation ◽

Maternal Mrna ◽

Term Potentiation ◽

Body Patterning

SUMMARY Maternal mRNA translation is regulated in large part by cytoplasmic polyadenylation. This process, which occurs in both vertebrates and invertebrates, is essential for meiosis and body patterning. In spite of the evolutionary conservation of cytoplasmic polyadenylation, many of the cis elements and trans-acting factors appear to have some species specificity. With the recent isolation and cloning of factors involved in both poly(A) elongation and deadenylation, the underlying biochemistry of these reactions is beginning to be elucidated. In addition to early development, cytoplasmic polyadenylation is now known to occur in the adult brain, and there is circumstantial evidence that this process occurs at synapses, where it could mediate the long-lasting phase of long-term potentiation, which is probably the basis of learning and memory. Finally, there may be multiple mechanisms by which polyadenylation promotes translation. Important questions yet to be answered in the field of cytoplasmic polyadenylation are addressed.

Download Full-text