Translation imaging of single mRNAs in established cell lines and primary cultured neurons

Methods. 2019 Jun 1;162-163:12-22. doi: 10.1016/j.ymeth.2019.03.021. Epub 2019 Mar 21.

Abstract

The central dogma of molecular biology reaches a crescendo at its final step: the translation of an mRNA into its corresponding protein product. This process is highly regulated both spatially and temporally, requiring techniques to interrogate the subcellular translational status of mRNAs in both living and fixed cells. Single-molecule imaging of nascent peptides (SINAPs) and related techniques allow us to study this fundamental process for single mRNAs in live cells. These techniques enable researchers to address previously intractable questions in the central dogma, such as the origin of stochastic translational control and the role of local translation in highly polarized cells. In this review, we present the methodology and the theoretical framework for conducting studies using SINAPs in both established cell lines and primary cultured neurons.

Keywords: Fluorescence microscopy; Single-molecule; Translation; mRNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • Capsid Proteins / chemistry
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism
  • Cell Line, Tumor
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Hippocampus / cytology
  • Humans
  • Intravital Microscopy / methods*
  • Levivirus / genetics
  • Mice
  • Neurons / metabolism*
  • Peptides / genetics
  • Peptides / metabolism
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Primary Cell Culture / methods
  • Protein Biosynthesis
  • RNA, Messenger / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Single Molecule Imaging / methods*
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / metabolism
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • Capsid Proteins
  • Peptides
  • Plant Proteins
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • scFV green fluorescent protein, recombinant
  • Green Fluorescent Proteins
  • Ubiquitin-Protein Ligases