Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes

Fujita, Hirotaka; Oikawa, Ryota; Hayakawa, Mayu; Tomoike, Fumiaki; Kimura, Yasuaki; Okuno, Hiroyuki; Hatashita, Yoshiki; Oliveros, Carolina Fiallos; Bito, Haruhiko; Ohshima, Toshio; Tsuneda, Satoshi; Abe, Hiroshi; Inoue, Takafumi

doi:10.1074/jbc.ra119.010921

Cited by 7 publications

(6 citation statements)

References 65 publications

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“…As the reported mRNA half-life range in the multi-hour scale (Schwanhäusser et al 2011, Tushev et al 2018), mRNA degradation was not considered in our model. Generally, our compiled distribution of simulated mRNAs' distances follows an exponential distribution with the highest mRNA density closest to the soma, as reported previously (Fonkeu et al 2019, Fujita et al 2020). The mRNAs' furthest distance traveled increased over time, reaching ~ 100 μm within 20 minutes, consistent with previous reports that newly transcribed mRNAs localize to the dendrites on the timescales of hours (Akbalik et al 2017).…”

Section: Simulation Of Passive Transport In 1dsupporting

confidence: 82%

Characterizing the Spatial Distribution of Dendritic RNA at Single Molecule Resolution

Kim,

Rosario,

Mendoza

et al. 2024

Preprint

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Neurons possess highly polarized morphology that require intricate molecular organization, partly facilitated by RNA localization. By localizing specific mRNA, neurons can modulate synaptic features through local translation and subsequent modification of protein concentrations in response to stimuli. The resulting activity-dependent modifications are essential for synaptic plasticity, and consequently, fundamental for learning and memory. Consequently, high-resolution characterization of the spatial distribution of dendritic transcripts and the spatial relationship across transcripts is critical for understanding the pathways and mechanisms underlying synaptic plasticity. In this study, we characterize the spatial distribution of six previously uncharacterized genes (Adap2,Colec12,Dtx3L,Kif5c,Nsmf,Pde2a) within the dendrites at a sub-micrometer scale, using single-molecule fluorescence in situ hybridization (smFISH). We found that spatial distributions of dendritically localized mRNA depended on both dendrite morphology and gene identity that cannot be recreated by diffusion alone, suggesting involvement of active mechanisms. Furthermore, our analysis reveals that dendritically localized mRNAs are likely co-transported and organized into clusters at larger spatial scales, indicating a more complex organization of mRNA within dendrites.

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Section: Simulation Of Passive Transport In 1dsupporting

confidence: 82%

Characterizing the Spatial Distribution of Dendritic RNA at Single Molecule Resolution

Kim,

Rosario,

Mendoza

et al. 2024

Preprint

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“…264 It has been used thus far, among other applications, to determine the composition of RNPs, 265 to analyze the diffusion and hybridization rates of different oligonucleotide populations, 266 and to measure mRNA dynamics in living neurons. 267…”

Section: Methods For Determining Rna Structures Involved In Molecular...mentioning

confidence: 99%

“…Despite these technical limitations, FCS serves as a valuable tool for analyzing molecular diffusion through the measurement of fluorescence intensity fluctuations and has found widespread use in the investigation of various biological systems, including MLOs and phase-separated bodies . It has been used thus far, among other applications, to determine the composition of RNPs, to analyze the diffusion and hybridization rates of different oligonucleotide populations, and to measure mRNA dynamics in living neurons …”

Section: Methods To Study Rna Crowdingmentioning

confidence: 99%

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding

Zacco,

Broglia,

Kurihara

et al. 2024

Chem. Rev.

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This comprehensive Review delves into the chemical principles governing RNA-mediated crowding events, commonly referred to as granules or biological condensates. We explore the pivotal role played by RNA sequence, structure, and chemical modifications in these processes, uncovering their correlation with crowding phenomena under physiological conditions. Additionally, we investigate instances where crowding deviates from its intended function, leading to pathological consequences. By deepening our understanding of the delicate balance that governs molecular crowding driven by RNA and its implications for cellular homeostasis, we aim to shed light on this intriguing area of research. Our exploration extends to the methodologies employed to decipher the composition and structural intricacies of RNA granules, offering a comprehensive overview of the techniques used to characterize them, including relevant computational approaches. Through two detailed examples highlighting the significance of noncoding RNAs, NEAT1 and XIST, in the formation of phase-separated assemblies and their influence on the cellular landscape, we emphasize their crucial role in cellular organization and function. By elucidating the chemical underpinnings of RNAmediated molecular crowding, investigating the role of modifications, structures, and composition of RNA granules, and exploring both physiological and aberrant phase separation phenomena, this Review provides a multifaceted understanding of the intriguing world of RNA-mediated biological condensates.

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“…Fujita et al used reduction-triggered fluorescent probes combined with fluorescence correlation spectroscopy and observed in real time that Arc mRNA was produced after synaptic activation [86]. Analysis of observations confirmed that free diffusion is not enough to transport RNAs of sizes close to Arc mRNA.…”

Section: Arc Mrna Trafficking Within Neuronsmentioning

confidence: 99%

mRNA Trafficking in the Nervous System: A Key Mechanism of the Involvement of Activity-Regulated Cytoskeleton-Associated Protein (Arc) in Synaptic Plasticity

et al. 2021

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Synaptic activity mediates information storage and memory consolidation in the brain and requires a fast de novo synthesis of mRNAs in the nucleus and proteins in synapses. Intracellular localization of a protein can be achieved by mRNA trafficking and localized translation. Activity-regulated cytoskeleton-associated protein (Arc) is a master regulator of synaptic plasticity and plays an important role in controlling large signaling networks implicated in learning, memory consolidation, and behavior. Transcription of the Arc gene may be induced by a short behavioral event, resulting in synaptic activation. Arc mRNA is exported into the cytoplasm and can be trafficked into the dendrite of an activated synapse where it is docked and translated. The structure of Arc is similar to the viral GAG (group-specific antigen) protein, and phylogenic analysis suggests that Arc may originate from the family of Ty3/Gypsy retrotransposons. Therefore, Arc might evolve through “domestication” of retroviruses. Arc can form a capsid-like structure that encapsulates a retrovirus-like sentence in the 3 ′ -UTR (untranslated region) of Arc mRNA. Such complex can be loaded into extracellular vesicles and transported to other neurons or muscle cells carrying not only genetic information but also regulatory signals within neuronal networks. Therefore, Arc mRNA inter- and intramolecular trafficking is essential for the modulation of synaptic activity required for memory consolidation and cognitive functions. Recent studies with single-molecule imaging in live neurons confirmed and extended the role of Arc mRNA trafficking in synaptic plasticity.

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Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes

Cited by 7 publications

References 65 publications

Characterizing the Spatial Distribution of Dendritic RNA at Single Molecule Resolution

Characterizing the Spatial Distribution of Dendritic RNA at Single Molecule Resolution

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding

mRNA Trafficking in the Nervous System: A Key Mechanism of the Involvement of Activity-Regulated Cytoskeleton-Associated Protein (Arc) in Synaptic Plasticity

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