Single-molecule insights into mRNA dynamics in neurons

Abstract

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.

Keywords: RNA localization; local translation; mRNA tracking; smFISH.

Figure 1. mRNA distribution in neurons varies widely

a. Single molecule FISH of endogenous β-actin mRNA in cultured primary hippocampal neurons demonstrates that the mRNA localizes along neurites. b. SmFISH of CaMKIIα mRNA in cultured neurons. c. SmFISH of Spinophillin mRNA localized in neurites. d. FISH using dT sequences complementary to Poly(A) demonstrate the total amount of mRNA in dendrites. e. Visualization of endogenous GAPDH mRNA with smFISH shows that the mRNA is largely restricted to the soma. The mRNA signal was dilated by 2 pixels to aid viewing. The heat map represents the relative intensity. Bar, 10µm.

Figure 2. The life of a localized mRNA in a neuron

a. During, or shortly following synthesis, mRNAs are recognized and bound to RNA Binding Proteins (RBPs), which play a role in all aspects of the mRNAs travels and life. b. In neurons, a large proportion of mRNAs are actively transported. Although it remains unclear where precisely the mRNA binds motors, it is possible that assembly of the transport machinery occurs at or near the soma since many mRNAs can be seen actively transported in regions near the soma. c. Navigation of mRNAs through the arbor is a poorly understood process. Whether transport is random or biased towards specific branches or regions is unknown. Nevertheless, mRNA transport must account for the highly branched morphology of a neuron. d. mRNAs can be captured or stalled at specific regions in response to local activity. This may allow for the specific targeting of proteins to particular regions or synapses through local translation. e. mRNA imaging highlights the oscillatory behavior of actively transported mRNAs, reminiscent of random searching. This could possibly be due to multiple opposing motors associated with the mRNP, bipolar orientation of microtubules or alternating binding between microtubules and actin filaments. Future work will reveal more on how this behavior is regulated and how random it is.