Background

Recently, it has become possible to measure the translation dynamics of single mRNA molecules in live cells (Morisaki et al., 2016; Pichon et al., 2016; Wang et al., 2016; Wu et al., 2016; Yan et al., 2016). This technology allows for the quantitation of the heterogeneity of translation dynamics among mRNA (Morisaki and Stasevich, 2018). To image and quantitate single mRNA translation dynamics, fluorescent intrabodies such as Fab (Hayashi-Takanaka et al., 2011), scFv (Tanenbaum et al., 2014; Zhao et al., 2019), or nanobodies (Boersma et al., 2019) are required. These intrabodies bind and label repeated epitopes inserted at the N-terminus of a protein of interest. As the protein of interest is translated, the repeated epitopes emerge from the ribosome and are bound within seconds by the fluorescent intrabodies (Morisaki and Stasevich, 2018; Cialek et al., 2020). This strategy amplifies fluorescence within the translation sites at two levels: firstly, multiple fluorescent intrabodies can bind the repeated epitopes within a single nascent peptide chain at the same time; and secondly, multiple ribosomes can translate the mRNA in polysomes to produce multiple nascent peptide chains within a single translation site. These two levels of amplification produce bright fluorescent puncta that can be detected with single-molecule precision above the background using a sensitive fluorescence microscope. As ribosomes initiate, elongate, and terminate translation, the fluorescence intensity at individual translation sites fluctuates up or down, yielding insight into translation dynamics.

Over the last few years, complementary intrabodies and epitopes have been developed that make it possible to image translation in multiple colors at the same time. For example, our lab has developed anti-FLAG and anti-HA Fab that bind the classic FLAG and HA epitopes (Morisaki et al., 2016). More recently, we developed a genetically encodable scFv version of the anti-HA Fab that we call the anti-HA frankenbody (Zhao et al., 2019). Likewise, the Tanenbaum lab has developed the SunTag (SunTag epitopes + anti-SunTag scFv-GFP) and MoonTag (MoonTag epitopes + anti-MoonTag nanobody) systems (Tanenbaum et al., 2014; Boersma et al., 2019). Used together, these complementary imaging tools make it possible to compare the dynamics of different open reading frames (ORFs) at the single-molecule level within live cells.

Here, we describe how to use a 3-color bicistronic biosensor that we recently developed to compare translation kinetics at internal ribosomal entry sites (IRES) versus at the canonical 5’ cap (Figure 1a) (Koch et al., 2020). The biosensor encodes an MS2 tag to mark and track individual mRNA molecules (red) (Coulon et al., 2013; Pichon et al., 2020). When these mRNAs are expressed in cells, they light up in different colors depending on the nature of translation initiation. If translation is initiated at the cap, then FLAG epitopes are translated and bound by anti-FLAG Fab conjugated with Cy3 (green). If translation is initiated at the IRES, then SunTag epitopes are translated and bound by anti-SunTag scFv fused to eGFP (blue). This produces a variety of colorful puncta in cells that mark individual mRNA and their translation status (Figure 1a and 1c).

This basic imaging biosensor is unique because it enables quantitative comparisons between the translation dynamics of two ORFs at a spatial resolution on the tens-of-nanometers scale and temporal resolution on the sub-seconds scale. This is not possible with more traditional bulk assays that lack single-molecule resolution, including traditional luciferase and GFP reporters as well as bulk assays like western blotting. The biosensor is also versatile because a different IRES sequence can easily be inserted to study the translational dynamics of that IRES in diverse cell types and cellular conditions (Bornes Stéphanie et al., 2007; Firth and Brierley, 2012). Further, our 3-color assay can be extended to compare ORF translation dynamics on two separate mRNAs (Figure 1b). This can be achieved by simply splitting the bicistronic biosensor into two monocistronic biosensors, each encoding a probe/epitope pair and an MS2 tag. This setup is not only beneficial for comparing the translation dynamics of two different ORFs but also for fairly comparing different endogenous 5’ or 3’ UTRs or viral elements within single cells.

In this protocol, we focus mainly on the wet lab skills needed to express our biosensor in live human cells. In particular, since our biosensor fluoresces in three colors, multiple components need to be expressed within cells at appropriate levels. Here, we advocate the use of beadloading (McNeil and Warder, 1987; Hayashi-Takanaka et al., 2011) to achieve this with high efficiency and minimal effort. We describe how the various probes – including the MS2 coat protein used to label mRNA with an MS2 tag, Fab to label FLAG-tagged epitopes, and SunTag scFv to label SunTag epitopes – can be purified and loaded together in a single, simple step (Cialek et al., 2021; Morisaki et al., 2016; Koch et al., 2020). We find this approach to be very convenient, enabling the fine-tuning of probe levels, rapid testing, and combinatorial experimentation. Finally, we describe the basics of our imaging and analyses, including a specific test to verify translation, useful conditions for stressing cells while under the microscope, and basic codes to quantitate translation site intensities and distributions.

Figure 1. 3-color imaging of the translation of a single bicistronic or pair of monocistronic biosensors in live cells. a) Schematic illustrating a bicistronic 3-color single-molecule translation reporter. b) Schematic illustrating a 2-mRNA single-molecule translation reporter system. c) A 3-color bicistronic reporter (representative cell). Crops showing all types of translation are on the right panel.