Multi-Color Single-Molecule Imaging Uncovers Extensive Heterogeneity in mRNA Decoding

Abstract

Summary mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real time. We find that start site selection is largely stochastic but that the probability of using a particular start site differs among mRNA molecules and can be dynamically regulated over time. This study provides key insights into translation start site selection heterogeneity and provides a powerful toolbox to visualize complex translation dynamics.

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

“…We used transient transfection in HeLa cells as a model system to test the binding properties of these protein binders (Brauchle et al, 2014; Moutel et al, 2016; Vigano et al, 2018). We therefore generated mammalian expression constructs for the anti- GCN4 (SunTag) scFv (Tanenbaum et al, 2014), the anti-gp41(MoonTag) nanobody 2H10 (Boersma et al, 2019), the anti-HA scFvs frankenbodies (Zhao et al, 2019) and the anti-ALFA nanobody (Gotzke et al, 2019) fused to either sfGFP or mEGFP for intracellular visualization. All the binders were expressed under the control of the strong CMV promoter/enhancer (for a summary of binder constructs, see Figure 1).…”

“…We focused our study on short peptide tags for which specific, high-affinity binders, which are soluble and functional in the intracellular milieu have been characterized. Therefore, we selected the following systems: SunTag (Tanenbaum et al, 2014), MoonTag (Boersma et al, 2019), HA (Zhao et al, 2019) and ALFA (Gotzke et al, 2019). For other commonly used tags such as FLAG® (Hopp et al, 1988) or Myc (Evan et al, 1985), we are unaware of specific binders derived from the corresponding monoclonal antibodies that could perform as intrabodies (Fujiwara et al, 2002; Marschall et al, 2015; Moutel et al, 2016; Worn et al, 2000).…”

Reflections on the use of protein binders to study protein function in developmental biology

“…Therefore, the use of recently reported intrabodies against small tags, such as SunTag (19aa) Degraded in absence of the antigen (GFP) (Tang et al, 2016) Lysine-less VHH4 (GFP, YFP, Venus) VHH4 in which lysines have been substituted for Arginines (Daniel et al, 2018) mCherry VHH (Fridy et al, 2014) MoonTag (gp41, 15aa linear epitope) (Boersma et al, 2019) BC2 VHH (BC2 tag, 12aa) Not tested as intrabody. (Braun et al, 2016) NbALFA (ALFAtag, 15aa) (Götzke et al, 2019) Darpin 3G86.32 Anti-GFP DARPin (GFP) (Brauchle et al, 2014) 2 m22 Anti-mCherry DARPin (mCherry) (Brauchle et al, 2014) E11 and G01 Anti-TFP DARPins (TFP) (Vigano et al, 2018) scFv HA frankenbody (HA, 9aa linear epitope) (Zhao et al, 2018) Suntag (GCN4 v4, 19aa linear epitope) Aggregation at high levels unless fused to sfGFP-GB1 scaffold (Tanenbaum, Gilbert, Qi, Weissman, & Vale, 2014) BGP7 ScFv (BGP7, 7aa linear epitope) (Lim, Ichinose, Shinoda, & Ueda, 2007;Wongso, Dong, Ueda, & Kitaguchi, 2017) (15aa), promises to be a great addition to the protein binder toolbox (Boersma et al, 2019;Gotzke et al, 2019;Tanenbaum et al, 2014;Zhao et al, 2018). In most cases, the short peptide tags recognized by these intrabodies has been used in multimerized form such as to recognize single protein particles; whether these short tags also work well when integrated as a single tag remains to be tested.…”

“…In addition to SunTag, intrabodies against the HA-tag (9 aa (Zhao et al, 2018)), MoonTag, a 15aa peptide from the HIV envelop protein complex subunit gp41 (Boersma et al, 2019), and ALFAtag (15aa, Götzke et al, 2019), have recently been validated. The availability of an array of intrabodies against small tags opens the exciting possibility of tagging the POI with different tags and following the dynamics of the POI via one tag at the same time as manipulating the POI via the other tag.…”

“…Furthermore, the POI and chromobody must be co-localized upon binding (e.g., by immunostaining of the POI) (Panza et al, 2015;Son et al, 2016). To quantify the level of POI, it might be useful to determine the background fluorescence level and subtract it from the experimental data (Boersma et al, 2019).…”