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:       TU Delft
  • Date:             11-05-2016
  • Author:         Katja Slangewal

The fate and function of a cell depend among others on the protein expression control. The lifetime of mRNA is approximately 10 hours and the lifetime of proteins is approximately 20 hours. This means that every protein is renewed each day. These numbers stress the importance of ribosomes. This is why Marvin Tanenbaum and his group explore the ribosome thoroughly. They want to know the speed, variability, noise and error rate of this complex molecular machine.

The variation in gene expression can be seen in western blots of several proteins over time. There is a large difference in expression of certain proteins between cells in the G1 phase and cells in the M phase for instance. However, these experiments involve a lot of averaging over different cells. Single cells can behave very different. They are all stochastic processes, so even single cells differ a lot over time. The question is: how do you assure, on single cell basis that a cell behaves as it is supposed to?

Before it is possible to answer this question, it is important to know how you observe single cell gene expression. The first approach included using GFP as a tag. Unfortunately a problem occurred. This problem was the maturation time of GFP. It takes approximately 5-60 minutes before GFP becomes visible. This time is much larger than the translation time, which means you cannot see the protein being translated. Additionally GFP has a very weak signal. So Marvin and his colleagues searched for more options.

They found a solution in using secondary tags. They made sure that GFP was abound and could bind to the SunTag (SuperNova tag) added to the protein. This technique enabled to see the translation from the start. The SunTag could bind many GFP’s causing very bright blobs. Since there are many ribosomes at each mRNA strand, it was not yet possible to track a single mRNA strand.
Graphical abstract.ai

Figure 1: The SunTag labeled with GFP. This tag helped the Tanenbaum group to see single ribosomes and to see the transcriptional activation [1].

Next, they added not only a SunTag to the gene, but also a mcherry tag. The last one was added after the stop-codon. So now it was possible to follow a single mRNA molecule. Then the task was to find the ribosome speed. This was done by blocking translation at once. No new ribosomes were able to bind to the mRNA strand and the remaining ones were slowly removed. The decrease in GFP signal allowed calculating the ribosome rate, which appeared to be 2-5 codons per second. Marvin has the opinion that this is a large variation, since the speed was measured over ~1000 steps. The variation could be because ribosomes get stuck sometimes. However, this needs to be validated with future research.

Finally, Marvin showed how they accomplished to follow a single ribosome. They used a clever trick for that. By adding a certain structure at the start of a sequence the mRNA forms a structure. Many ribosomes fall off after bumping in this structure, but rarely a ribosome is able to stay on the mRNA. If that is the case, you can observe a single ribosome.

I think Marvin gave a clear talk. The structure was quite nice. He clearly stressed the problems they encountered and the solutions they found for them. It was nice to see the progress in his research. He started his talk very calm and I was afraid it would get boring. Luckily Marvin became more and more enthusiastic as he went on with the talk. All together I liked the talk and I learned new things, like the fact that GFP has a maturation time.

[1] Tanenbaum M.E., Gilbert L.A., Qi L.S., Weissman J.S., Vale R.D. (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:3 635-646


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