Abstract
Transcription and post-transcriptional regulations are critical in gene expression. To study the spatiotemporal regulation of RNA inside a cell, techniques for high-resolution imaging of RNA have been developed. In this chapter, we describe RNA fluorescent labeling methods using MS2 and PP7 systems to detect single RNA molecules in live neurons. We use hippocampal neurons cultured from knock-in mouse models in which β-actin or Arc mRNAs are tagged with MS2 or PP7 stem-loops. Adeno-associated virus (AAV) or lentiviral vectors are used to express MS2 or PP7 capsid proteins fused with GFP in those neurons. Then, GFP-labeled RNAs in live neurons can be detected by epifluorescence microscopy, and their moving pathways can be analyzed using single-particle tracking software. For these processes, we introduce protocols for neuron culture, transfection, imaging, and particle tracking methods.
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References
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25(2):169–193. https://doi.org/10.1677/jme.0.0250169
Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A 74(12):5350–5354. https://doi.org/10.1073/pnas.74.12.5350
Femino A, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280(5363):585–590. https://doi.org/10.1126/science.280.5363.585
Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. https://doi.org/10.1038/nmeth.1253
Moon HC, Lee BH, Lim K, Son JS, Song MS, Park HY (2016) Tracking single mRNA molecules in live cells. J Phys D Appl Phys 49(23). https://doi.org/10.1088/0022-3727/49/23/233001
Kim SH, Vieira M, Shim JY, Choi H, Park HY (2018) Recent progress in single-molecule studies of mRNA localization in vivo. RNA Biol:1–11. https://doi.org/10.1080/15476286.2018.1536592
Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2(4):437–445
Chao JA, Patskovsky Y, Almo SC, Singer RH (2008) Structural basis for the coevolution of a viral RNA-protein complex. Nat Struct Mol Biol 15(1):103–105. https://doi.org/10.1038/nsmb1327
Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, Singer RH (2011) A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods 8(2):165–170. https://doi.org/10.1038/Nmeth.1551
Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, Singer RH (2014) Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science 343(6169):422–424. https://doi.org/10.1126/science.1239200
Das S, Moon HC, Singer RH, Park HY (2018) A transgenic mouse for imaging activity-dependent dynamics of endogenous Arc mRNA in live neurons. Sci Adv 4(6):eaar3448. https://doi.org/10.1126/sciadv.aar3448
Ploski JE, Pierre VJ, Smucny J, Park K, Monsey MS, Overeem KA, Schafe GE (2008) The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is required for memory consolidation of pavlovian fear conditioning in the lateral amygdala. J Neurosci 28(47):12383–12395. https://doi.org/10.1523/Jneurosci.1662-08.2008
Park HY, Buxbaum AR, Singer RH (2010) Single mRNA tracking in live cells. Method Enzymol 472:387–406. https://doi.org/10.1016/S0076-6879(10)72003-6
Park HY, Trcek T, Wells AL, Chao JA, Singer RH (2012) An unbiased analysis method to quantify mRNA localization reveals its correlation with cell motility. Cell Rep 1(2):179–184. https://doi.org/10.1016/j.celrep.2011.12.009
Song MS, Moon HC, Jeon JH, Park HY (2018) Neuronal messenger ribonucleoprotein transport follows an aging Lévy walk. Nat Commun 9(1):344. https://doi.org/10.1038/s41467-017-02700-z
Wu B, Chao JA, Singer RH (2012) Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys J 102(12):2936–2944. https://doi.org/10.1016/j.bpj.2012.05.017
Lee BH, Park HY (2018) HybTrack: A hybrid single particle tracking software using manual and automatic detection of dim signals. Sci Rep 8(1):212. https://doi.org/10.1038/s41598-017-18569-3
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This work was supported by the Creative-Pioneering Researchers Program through Seoul National University.
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Shim, J.Y., Lee, B.H., Park, H.Y. (2019). Visualization of Single mRNAs in Live Neurons. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 2038. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9674-2_4
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DOI: https://doi.org/10.1007/978-1-4939-9674-2_4
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