Förster resonance energy transfer (FRET) microscopy is a powerful technique routinely used to monitor interactions between biomolecules. Here, we focus on the techniques that are used for investigating the structure and interactions of nucleic acids (NAs). We present a brief overview of the most commonly used FRET microscopy techniques, their advantages and drawbacks. We list experimental approaches recently used for either in vitro or in vivo studies. Next, we summarize how FRET contributed to the understanding of pre-mRNA splicing and spliceosome assembly.

• MeSH
• Alternative Splicing MeSH
• Microscopy, Fluorescence * MeSH
• Molecular Conformation MeSH
• Nucleoproteins metabolism   MeSH
• Nucleic Acids chemistry   genetics   metabolism   MeSH
• RNA Precursors chemistry   genetics   metabolism   MeSH
• Fluorescence Resonance Energy Transfer * MeSH
• RNA Splicing * MeSH
• Spliceosomes genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Nucleoproteins MeSH
• Nucleic Acids MeSH
• RNA Precursors MeSH

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

• MeSH
• Cell Nucleus metabolism   MeSH
• Cell Line MeSH
• Spectrometry, Fluorescence MeSH
• Fluorescence Recovery After Photobleaching MeSH
• HeLa Cells MeSH
• Kinetics MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• RNA Precursors metabolism   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   physiology   MeSH
• RNA Splicing physiology   MeSH
• Spliceosomes metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Messenger MeSH
• RNA Precursors MeSH
• Ribonucleoproteins, Small Nuclear MeSH