Adina R. Buxbaum

In the right place at the right time: visualizing and understanding mRNA localization

The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.

Single β-Actin mRNA Detection in Neurons Reveals a Mechanism for Regulating Its Translatability

The physical manifestation of learning and memory formation in the brain can be expressed by strengthening or weakening of synaptic connections through morphological changes. Local actin remodeling underlies some forms of plasticity and may be facilitated by local β-actin synthesis, but dynamic information is lacking. In this work, we use single-molecule in situ hybridization to demonstrate that dendritic β-actin messenger RNA (mRNA) and ribosomes are in a masked, neuron-specific form. Chemically induced long-term potentiation prompts transient mRNA unmasking, which depends on factors active during synaptic activity. Ribosomes and single β-actin mRNA motility increase after stimulation, indicative of release from complexes. Hence, the single-molecule assays we developed allow for the quantification of activity-induced unmasking and availability for active translation. Further, our work demonstrates that β-actin mRNA and ribosomes are in a masked state that is alleviated by stimulation. Imaging of β-actin messenger RNA (mRNA) in neurons reveals transient alteration of mRNA availability during synaptic plasticity. [Also see Perspective by Akbalik and Schuman] Observing the Messenger In order to elucidate the dynamics of individual components in the cell, single-molecule technologies are being developed (see the Perspective by Akbalik and Schuman). Park et al. (p. 422) used a mouse expressing fluorescent β-actin messenger RNAs (mRNAs) to visualize mRNA movements in living cells and tissues. Buxbaum et al. (p. 419) showed that neurons contain β-actin mRNAs and ribosomes packaged in a dense structure, impenetrable by oligonucleotide probes. This effectively masks the mRNAs until neuronal stimulation exposes the mRNA and ribosomes briefly, presumably reflecting the local stimulation and translation involved, for example, in the generation of memories.

Single mRNA tracking in live cells.

Asymmetric distribution of mRNA is a prevalent phenomenon observed in diverse cell types. The posttranscriptional movement and localization of mRNA provides an important mechanism to target certain proteins to specific cytoplasmic regions of their function. Recent technical advances have enabled real-time visualization of single mRNA molecules in living cells. Studies analyzing the motion of individual mRNAs have shed light on the complex RNA transport system. This chapter presents an overview of general approaches for single particle tracking and some methodologies that are used for single mRNA detection.

mRNA on the Move: The Road to Its Biological Destiny *□

Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades, evidence has mounted as to the importance of localization elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview, we focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement.

Single-molecule insights into mRNA dynamics in neurons

Targeting of mRNAs to neuronal dendrites and axons plays an integral role in intracellular signaling, development, and synaptic plasticity. Single-molecule imaging of mRNAs in neurons and brain tissue has led to enhanced understanding of mRNA dynamics. Here we discuss aspects of mRNA regulation as revealed by single-molecule detection, which has led to quantitative analyses of mRNA diversity, localization, transport, and translation. These exciting new discoveries propel our understanding of the life of an mRNA in a neuron and how its activity is regulated at the single-molecule level.

Quantifying Protein-mRNA Interactions in Single Live Cells

Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to β-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with β-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.

Glutamate-induced RNA localization and translation in neurons

Significance Local translation in dendrites of neurons has been shown to be important for neuronal function and synaptic biology. We imaged changes in the localization of β-actin mRNA and protein in dendritic spines. Our results showed that activating specific synapses can drive changes in the localization of endogenous mRNA and the translation of reporter RNA in dendrites of hippocampal neurons. Enhancing our understanding of the spatial and temporal kinetics of mRNA localization in dendrites informs local protein synthesis in neurons. These results provide direct evidence of protein synthesis away from the soma and allow us to determine how the kinetics of mRNA localization and translation could influence synaptic physiology and plasticity. Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapse-specific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single β-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcode-binding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of β-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.

Erratum: In the right place at the right time: Visualizing and understanding mRNA localization (Nature Reviews Molecular Cell Biology (2015) 16 (95-109))

In the right place at the right time: visualizing and understanding mRNA localization Adina R. Buxbaum, Gal Haimovich and Robert H. Singer Nature Reviews Molecular Cell Biology 16, 95–109 (2015) In the original article, the two reference citations in the following sentence were incorrect: “... conversely, only 600–800 mammalian RBPs have been recognized so far (REFS 88, 89; see the RBP database) ...”. The correct references have now been added to the online version of this articleas as references 177 and 178.