Dynamics of translation of single mRNA molecules in vivo

Speaker: Marvin Tanenbaum

Department: Hubrecht Institute, Utrecht

Subject: Dynamics of translation of single mRNA molecules in vivo

Location: Bionanoscience Department, TU Delft

Date: 24-03-2017

Author: Mirte Golverdingen

Marvin Tanenbaum is interested in the control and principle of gene expression. Gene expression is not static, it is, instead, very dynamic, as seen during the cell cycle. It has a role in the regulation of all biology and this is visualized when looking at the influence of gene expression on the central dogma. Gene expression regulates transcription and translation in the cells. However, because the cell cytoplasma is so complex and chaotic, single molecule visualization of this process is hard. So, how do we get from the chaos to the pattern each cell has?

During transcription, translation and the path of mRNA to the cytoplasm, gene expression is regulated by a large number of molecules.  These processes are most of the time studied by using GFP tracking. Twenty years ago, Singer et al. tracked mRNA for the first time by using fluorescent proteins that were attached to hairpin loops. However, for real-time visualization of translation, GFP tagging does not work because of the very weak signal of single molecules. Moreover, GFP has a ‘maturation’ time that takes very long in comparison with the translation time.

Tanenbaum started to label the ribosomes indirectly by using a GFP -Antibody-Small-peptide.  In this way, more fluorescent proteins can bind to the peptide, creating a stronger signal. However, this method was very hard to implicate. Therefore, they used a chain of GFP proteins attached to a SunTag protein that can attach to a protein label. This sequence of the protein label was translated and the resulting mRNA’s were labeled with a red mCherry dye that was attached to the hairpins of the mRNA (see Figure 1A). This system is 15~20 fold brighter than a single GFP protein, and has a good affinity for the targets.

After developing this imaging system, they applied it to obtain direct observation of translation by using the developed SunTag. When the mCherry red dots also shows green dots, translation is happening.To test if there was really translation going on, puromycin (a translation inhibitor) was added. After this addition, no green dots were seen anymore. Therefore, the combination of red and green dots really shows real-time translation(see Figure 1C). However, because translation is a very dynamic process, tracking a single mRNA was very hard. Therefore, they attached the mRNA to the plasma membrane. In this way, a single mRNA could be tracked during his whole life time (see Figure 1D and 1E).

Now real-life imaging of translation was possible, the quantitative nature of the translation could be measured. They counted the number of the ribosomes on the translation site, resulting in 20 ribosomes on each translation site. Moreover, to calculate the speed of the ribosomes, they added the translation initiation inhibitor harringtonine. In this way, you can measure the rate of the disappearing ribosomes. They calculated that ribosomes have a translation speed of 3-5 codons/s.

By adding an untranslated region (UTR), they could see a single ribosome decode a mRNA, because by using an UTR only once in a while a ribosome will bind to the mRNA. They observed only small flashes of green light after adding UTR. So, in this way they could observe single ribosomes that read RNA’s. Moreover, they observed a mRNA cycle between a translating and non-translating state. However, they have no idea yet why this cycle is there.

In conclusion, Tanenbaum visualized real-time translation of single mRNA’s for the first time by using the SunTag system. They could quantify the number of ribosomes on the translation site and the ribosome translation speed by using this method. Moreover, they were able to visualize a single ribosome decoding a mRNA. I think that this technique is an important contribution to the research of gene expression. Moreover, wide applications of the assay can contribute to the understanding of the complex process of translation. Being able to see the ‘abstract’ process of translation in real time in a cell is overall very cool.

Honours 8

Figure 1: Fluorescence Labeling of Nascent Chains to Visualize Translation of Single mRNA Molecules.

(A) Schematic of nascent polypeptide labeling using the SunTag system and mRNA labeling (A) and membrane tethering (D) using the PP7 system.

(B) A mCherry-SunTag24x reporter gene was co-transfected with either GFP or scFv-GFP, and the expression of the SunTag24x-mCherry reporter was determined by FACS (Experimental Procedures). Binding of the scFv-GFP to the SunTag nascent chain did not detectably alter protein expression.

(C) A representative U2OS cell is shown expressing scFv-GFP, PP7-3xmCherry, and the translation reporter (SunTag24x-Kif18b-PP724x). Cytosolic translation sites (scFv-GFP) co-localize with mRNAs (PP7-3xmCherry). Ribosomes were dissociated from mRNA by addition of puromycin (right panel). Note that translation sites and mRNA do not perfectly overlap because of the brief time difference in acquiring GFP and mCherry images.

(D) Schematic of nascent polypeptide labeling and membrane tethering of the mRNA using the PP7 system.

(E) U2OS cells expressing scFv-GFP (green), PP7-2xmCherry-CAAX (red), and the translation reporter (SunTag24x-Kif18b-PP724x). A single time point of the cell (top panel) and a zoomed-in view from the white-boxed area containing a few mRNAs (lower) are shown.

Adapted from: Yan, Xiaowei et al. (2016) Dynamics of Translation of Single mRNA Molecules In Vivo. Cell , Volume 165 , Issue 4 , 976 – 989

 

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