Probing the Conformational State of mRNPs Using smFISH and SIM

Methods Mol Biol. 2021;2209:267-286. doi: 10.1007/978-1-0716-0935-4_17.

Abstract

mRNAs and lncRNAs assemble with RNA-binding proteins (RBPs) to form ribonucleoprotein complexes (RNPs ). The assembly of RNPs initiates co-transcriptionally, and their composition and organization is thought to change during the different steps of an RNP life cycle. Modulation of RNP structural organization has been implicated in the regulation of different aspects of RNA metabolism, including establishing interactions between the 5' and 3' ends in regulating mRNA translation and turnover. In this chapter, we describe a single-molecule microscopy approach that combines fluorescent RNA in situ hybridization (smFISH) and structured illumination microscopy (SIM ) and allows to measure different aspects of RNP organization in cells, including distances between different regions within individual mRNAs, as well as the overall compaction state of RNAs in different subcellular compartments and environmental conditions. Moreover, we describe a detailed workflow required for image registration and analysis that allows determining distances at sub-diffraction resolution.

Keywords: RNA organization; RNA structure; Single-molecule resolution microscopy; Structured illumination microscopy; lncRNAs; mRNA; mRNPs; smFISH.

MeSH terms

  • In Situ Hybridization, Fluorescence / methods*
  • Nucleic Acid Conformation
  • RNA, Long Noncoding / chemistry*
  • RNA, Messenger / chemistry*
  • RNA-Binding Proteins / chemistry*
  • Ribonucleoproteins / chemistry*
  • Single Molecule Imaging / methods*

Substances

  • RNA, Long Noncoding
  • RNA, Messenger
  • RNA-Binding Proteins
  • Ribonucleoproteins
  • messenger ribonucleoprotein