Skip to main content
SpringerLink
Log in

Measuring miRNA Mediated Translational Regulation with Live Cell Imaging

  • Protocol
  • First Online:
MicroRNA Technologies

Part of the book series: Neuromethods ((NM,volume 128))

  • 649 Accesses

Abstract

Numerous microRNAs are detected in synaptic areas such as axons and dendrites. As accurate regulation of local protein synthesis can be crucial for the function of neurons, miRNAs can play very important roles for mRNA translation in the rather isolated cellular spaces such as synaptic area. However, due to the technical limitation, it is very difficult to measure efficiency of protein synthesis in the synaptic area with biochemical methods. Therefore, visualizing translation and measuring protein levels at the synaptic sites by imaging techniques can be a good alternative. Fluorescence recovery after photobleaching (FRAP) has been widely used to measure local protein synthesis rate in axons. This technique allows us to measure speed and efficiency of translation of certain mRNAs. We modified this method to measure how miRNAs influence on local synthesis of proteins in neurons.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Schratt GM, Tuebing F, Nigh EA, Kane CG, Sabatini ME, Kiebler M, Greenberg ME (2006) A brain-specific microRNA regulates dendritic spine development. Nature 439(7074):283–289. doi:10.1038/nature04367

    Article  CAS  PubMed  Google Scholar 

  2. Kye MJ, Liu T, Levy SF, Xu NL, Groves BB, Bonneau R, Lao K, Kosik KS (2007) Somatodendritic microRNAs identified by laser capture and multiplex RT-PCR. RNA 13(8):1224–1234. doi:10.1261/rna.480407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Natera-Naranjo O, Aschrafi A, Gioio AE, Kaplan BB (2010) Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons. RNA 16(8):1516–1529. doi:10.1261/rna.1833310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Holt CE, Schuman EM (2013) The central dogma decentralized: new perspectives on RNA function and local translation in neurons. Neuron 80(3):648–657. doi:10.1016/j.neuron.2013.10.036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Dieterich DC, Hodas JJ, Gouzer G, Shadrin IY, Ngo JT, Triller A, Tirrell DA, Schuman EM (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 13(7):897–905. doi:10.1038/nn.2580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Taylor AM, Dieterich DC, Ito HT, Kim SA, Schuman EM (2010) Microfluidic local perfusion chambers for the visualization and manipulation of synapses. Neuron 66(1):57–68. doi:10.1016/j.neuron.2010.03.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wu B, Eliscovich C, Yoon YJ, Singer RH (2016) Translation dynamics of single mRNAs in live cells and neurons. Science 352(6292):1430–1435. doi:10.1126/science.aaf1084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Vuppalanchi D, Coleman J, Yoo S, Merianda TT, Yadhati AG, Hossain J, Blesch A, Willis DE, Twiss JL (2010) Conserved 3′-untranslated region sequences direct subcellular localization of chaperone protein mRNAs in neurons. J Biol Chem 285(23):18025–18038. doi:10.1074/jbc.M109.061333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yoon YJ, Wu B, Buxbaum AR, Das S, Tsai A, English BP, Grimm JB, Lavis LD, Singer RH (2016) Glutamate-induced RNA localization and translation in neurons. Proc Natl Acad Sci U S A 113(44):E6877–E6886. doi:10.1073/pnas.1614267113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Niehues S, Bussmann J, Steffes G, Erdmann I, Kohrer C, Sun L, Wagner M, Schafer K, Wang G, Koerdt SN, Stum M, Jaiswal S, RajBhandary UL, Thomas U, Aberle H, Burgess RW, Yang XL, Dieterich D, Storkebaum E (2015) Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases. Nat Commun 6:7520. doi:10.1038/ncomms8520

    Article  PubMed  PubMed Central  Google Scholar 

  11. Akten B, Kye MJ, Hao le T, Wertz MH, Singh S, Nie D, Huang J, Merianda TT, Twiss JL, Beattie CE, Steen JA, Sahin M (2011) Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits. Proc Natl Acad Sci U S A 108(25):10337–10342. 1104928108 [pii]. doi:10.1073/pnas.1104928108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rathod R, Havlicek S, Frank N, Blum R, Sendtner M (2012) Laminin induced local axonal translation of beta-actin mRNA is impaired in SMN-deficient motoneurons. Histochem Cell Biol 138(5):737–748. doi:10.1007/s00418-012-0989-1

    Article  CAS  PubMed  Google Scholar 

  13. Nie D, Di Nardo A, Han JM, Baharanyi H, Kramvis I, Huynh T, Dabora S, Codeluppi S, Pandolfi PP, Pasquale EB, Sahin M (2010) Tsc2-Rheb signaling regulates EphA-mediated axon guidance. Nat Neurosci 13(2):163–172. doi:10.1038/nn.2477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Spinelli KJ, Taylor JK, Osterberg VR, Churchill MJ, Pollock E, Moore C, Meshul CK, Unni VK (2014) Presynaptic alpha-synuclein aggregation in a mouse model of Parkinson’s disease. J Neurosci 34(6):2037–2050. doi:10.1523/JNEUROSCI.2581-13.2014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Banerjee S, Neveu P, Kosik KS (2009) A coordinated local translational control point at the synapse involving relief from silencing and MOV10 degradation. Neuron 64(6):871–884. doi:10.1016/j.neuron.2009.11.023

    Article  CAS  PubMed  Google Scholar 

  16. Kye MJ, Niederst ED, Wertz MH, Goncalves Ido C, Akten B, Dover KZ, Peters M, Riessland M, Neveu P, Wirth B, Kosik KS, Sardi SP, Monani UR, Passini MA, Sahin M (2014) SMN regulates axonal local translation via miR-183/mTOR pathway. Hum Mol Genet 23(23):6318–6331. doi:10.1093/hmg/ddu350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Risbud RM, Porter BE (2013) Changes in microRNA expression in the whole hippocampus and hippocampal synaptoneurosome fraction following pilocarpine induced status epilepticus. PLoS One 8(1):e53464. doi:10.1371/journal.pone.0053464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Boese AS, Saba R, Campbell K, Majer A, Medina S, Burton L, Booth TF, Chong P, Westmacott G, Dutta SM, Saba JA, Booth SA (2016) MicroRNA abundance is altered in synaptoneurosomes during prion disease. Mol Cell Neurosci 71:13–24. doi:10.1016/j.mcn.2015.12.001

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgement

This work is supported by Deutsche Forschungsgemeinschaft (KY-92/1), University of Cologne (Cologne Fortune), and Cure SMA.

Author information

Authors and Affiliations

  1. Institute of Human Genetics, University of Cologne, Kerpener Str. 34, 50931, Cologne, Germany

    Min Jeong Kye

Authors
  1. Min Jeong Kye
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Min Jeong Kye .

Editor information

Editors and Affiliations

  1. Institute of Human Genetics, University of Cologne, Cologne, Germany

    Min Jeong Kye

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kye, M.J. (2017). Measuring miRNA Mediated Translational Regulation with Live Cell Imaging. In: Kye, M. (eds) MicroRNA Technologies. Neuromethods, vol 128. Humana Press, New York, NY. https://doi.org/10.1007/7657_2017_2

Download citation

  • DOI: https://doi.org/10.1007/7657_2017_2

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7173-2

  • Online ISBN: 978-1-4939-7175-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation