Speaker: Marvin Tanenbaum
Department: Department of Bionanoscience
Subject: Real-time observation of translation of single mRNA molecules in live cells.
Location: Delft University of Technology
Date: 24 March 2017
Author: Romano van Genderen
The research by professor Tanenbaum was about the kinetics of translation. He started by talking about how many genes, about 20%, oscillate in gene expression during the cell cycle, setting off processes of division and differentiation. For this to happen, a very strong regulation of the genes is necessary. This happens on many levels, not only on the scale of transcription, but also translation. Translational regulation has been shown to be the most important process to regulate how much of a certain protein is produced. This regulation happens through miRNA and RNA binding proteins.
In order to more carefully study this regulation, it is needed to see the RNA translation in action. A relatively old method for visualizing RNA was developed by Singer labs. They build in a series of hairpins in the RNA. These hairpins are a binding site for a protein called MCP. This MCP protein has a GFP tag to allow for visualization.
Showing the translation product sounds like something that would not be too hard. Just let the ribosome translate the RNA for GFP and it should be visible. But a problem about this method is that you create a lot of background noise from the free-floating GFP. Another problem is that GFP needs some time to maturate, which takes longer than the translation. So they used a free-floating GFP-antibody complex that binds the protein that is being synthesized. This SunTag system they developed is very bright and allows for very good visualization.
Fig 1. An overview of the SunTag system. You can see them being used to look at proteins that are being synthesized (Tanenbaum et al, A Protein-Tagging System for Signal Amplification for Signal Amplification in Gene Expression and Fluorescence Imaging, Cell 159)
So now you can combine the two aforementioned approaches. First fuse some small peptides to your protein of interest. Then let green antibodies bind the peptides. At the same time, you attach mCherry to a newer form of MCP, PP7 and let it bind the RNA hairpins which are non-coding. Wherever you see yellow, active translation is going on. When PP7 is injected into the cytoplasm, it can now be used to follow a piece of mRNA from the export into the cytoplasm until its eventual degradation.
Using this technique a few experiments were done.
Firstly, they were able to count the number of ribosomes on a single piece of mRNA, showing that the average number was around 20.
Next, they showed the translation speed. Because when the translation would be very slow, the ribosome signal would slowly dim after adding a translation initiation inhibiting drug. If translation would be very fast, the signal would be lost immediately.
Other research was done on the regulation of translation. They did find that specific RNA cutting proteins only work when RNApol collides with them, “bumping them off”. The cutting step of this protein works automatically, but they need a collision with RNApol to release the protein and therefore the cut strand.
I did really enjoy the technical overview of the versatile SunTag procedure and the applications of it. I do expect even more findings to come from this method, especially if this method can also be expanded to work on DNA as well. This would be a good improvement of the method. But I continue to doubt if understanding of the kinetics of translation has any practical applications.