Imaging Single-mRNA Localization and Translation in Live Neurons

Abstract

Local protein synthesis mediates precise spatio-temporal regulation of gene expression for neuronal functions such as long-term plasticity, axon guidance and regeneration. To reveal the underlying mechanisms of local translation, it is crucial to understand mRNA transport, localization and translation in live neurons. Among various techniques for mRNA analysis, fluorescence microscopy has been widely used as the most direct method to study localization of mRNA. Live-cell imaging of single RNA molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein (mRNP) complexes in neurons. Here, we review recent advances in the study of mRNA localization and translation in live neurons using novel techniques for single-RNA imaging.

Keywords: MS2-GFP; RNA localization; live-cell imaging; local translation; single-molecule imaging.

Fig. 1

Schematic diagrams of local translation in dendritic spines and an axonal growth cone. (A) Illustration of mRNA localization in a neuron. (B) Localization of mRNA in dendritic spines. Translation of β-actin mRNA is repressed by ZBP1 during transport to the localization site. After translation, newly synthesized β-actin proteins accumulate at the periphery of the spines. Arc mRNA localizes selectively at active synapses and mediates local synthesis of Arc proteins, which play a role in AMPA receptor endocytosis. (C) Localization of mRNA in an axonal growth cone. BDNF and netrin-1 induce local translation of β-actin mRNA, which mediates growth cone turning toward extracellular cues. Sema3A induces local translation of MAP1B mRNA, which leads to growth cone collapse.

Fig. 2

Schematic diagrams of the TRICK and Suntag (or FLAG)-MBS systems. (A) Schematic of the TRICK system. Both PCP-GFP and MCP-RFP are bound to the untranslated mRNA. During translation, ribosomes read through the PBS sequence and knock off PCP-GFP from the mRNA. After the first round of translation, the mRNA is only labeled with RFP. (B) Suntag (or FLAG)-MBS system. Translation of RFP-labeled mRNA can be monitored by the fluorescence signal from scFV-GFP clustered on the nascent polypeptides of Suntag (or FLAG).