Probing nucleic acid interactions and pre-mRNA splicing by Förster Resonance Energy Transfer (FRET) microscopy

Abstract

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.

Figure 1

Förster resonance energy transfer (FRET) fundamentals: (a) Schematic representation of FRET. Excited donor (D) transfers its energy by a non-radiative process to the nearby acceptor (A), causing it to emit fluorescence. The distance between fluorophores should not exceed 10 nm; (b) The donor emission peak must overlap with the acceptor excitation spectrum. The grey area corresponds to the overlap region; (c) FRET efficiency as a function of the distance between donor and acceptor fluorophores (r_DA). Förster radius (R₀) of the ECFP-EYFP pair is 4.92 nm [11], R₀ for the Cy3–Cy5 pair is 5.6 nm according to the manufacturer (Amersham Biosciences).

Figure 2

An example of AB-FRET microscopy images of the CFP-YFP fusion protein chimera. Images were acquired with a laser scanning confocal microscope. FRET efficiency was calculated according to Equation 2 and is presented as a false-colored intensity image.

Figure 3

(a) Fluorescence lifetime imaging. As is illustrated by Jablonski diagrams, if a fluorophore (donor only) is exposed to the excitation light source, it starts to occupy higher electronic states, and energy is eventually released via vibrational relaxation and emission of fluorescence. The average time the fluorophore spends in the excited state corresponds to its fluorescence lifetime. However, in the presence of a suitable acceptor, the donor can transfer its energy to the acceptor fluorophore non-radiatively and thus return to the ground state. This depopulation of excited state causes shortening of the donor fluorescence lifetime. Model examples of fluorescence lifetime imaging curves are shown in grey boxes. During FLIM measurement in the time domain, the fluorophore is excited by a short pulse and its fluorescence emission is measured over time. If FRET occurs, the fluorescence lifetime decreases; (b) Model example of single-molecule FRET. Fluorescence emission after donor excitation is monitored separately in donor and acceptor channels, one molecule at a time. The resulting FRET efficiency is estimated from the D/A fluorescence ratio.

Figure 4

(a) Unspecific labeling of RNA by SytoxOrange. When the RNA-binding protein (RBP) tagged with yellow fluorescent protein (YFP) binds to RNA, FRET between YFP and SytoxOrange occurs and can be detected by FLIM; (b) RNA-binding mediated FRET technique. RNA with a putative binding site for RBP of interest and MS2 stem loop in close proximity is transcribed from the expression vector. MS2 coat protein tagged with YFP and CFP-tagged RBP are expressed in cells. If RBP binds to the putative binding site, fluorescent protein tags are positioned close to each other and FRET occurs; (c) RNA-binding FRET sensor. RBP that changes its conformation after binding to RNA of interest is tagged with a fluorescent donor and acceptor pair. When RBP is bound to RNA, the tags come close enough to each other to enable FRET; (d) Direct detection of FRET between RNA and protein. RNA of interest is Cy3-labeled by in vitro methods and then injected to cells. RBP of interest is stained with Cy5-labeled specific antibody. FRET between Cy3 and Cy5 is measured afterwards.

Figure 5

(a) Conserved sequence elements of metazoan pre-mRNAs. GU and AG consensus dinucleotides mark intron boundaries. There is a conserved adenosine residue, which serves as a branch point during formation of the intron lariat, and a pyrimidine-rich sequence (polypyrimidine tract) positioned between the branch point and conserved AG at the 3′SS; (b) Simplified scheme of the spliceosome assembly.

Figure 6

Schematic representation of the early stage of spliceosome assembly. 5′SS is bound by U1 snRNP. U2AF35 binds 3′SS, U2AF65 interacts with the polypyrimidine tract and stabilizes interaction of SF1 with the branch point. The U2AF heterodimer recruits U2 snRNP to the 3′SS.

Figure 7

Schematic representation of the spliceosomal core. Secondary structure model of the spliceosomal snRNAs U2–U6 from Saccharomyces cerevisiae with an intron bound. Watson-Crick base-pairs are depicted as lines, non-Watson-Crick pairs as circles. Adapted from [58].

Figure 8

Alternative splicing detection via in situ hybridization according to [61]. Cy3-labeled hybridization probe complementary to exon 1 is used together with either exon 2 or alternative intron complementary Cy5-labeled probe. FRET occurs when probes are bound to mRNA close to each other.