MS2 mRNA imaging in yeast – problem solved

Previously, on the story of MS2 labeling of mRNA in yeast: Roy Parker published a short letter to the editor, indicating that the MS2 system might cause accumulation of 3′ fragments. We wrote a response, showing that it is not always the case for endogenously expressed mRNAs, but it is exaggerated when over-expressed (Part 1)*. Later, Karsten Weis’s group confirmed Parker’s initial observation but their report still had some questions unanswered, and no solution to the problem; I was unhappy (Part 2).  Now, Evelina Tutucci and Maria Vera together with Jeet Biswas (all from Rob Singer’s lab) seem to have resolved the issue and solved the problem, with the development of the MBS version 6

I don’t have time to go to great details and my advice is  – go read the paper. In brief, Evelina & Maria first confirmed that there is a problem. This time, They did it the right way – by co-FISH to count full-length and fragments and look for accumulating aggergates. This was true not only with the original MBS sequence, but also with MBSv5.

(Jennifer Garcia from Parker lab – who started this whole thing with – contributed by doing Northern blots).

Then, they had a clever idea: when the MS2 system was originally developed, a mutation was introduced into the wild-type sequence, which increaed by 10-fold the affinity of the coat protein to the stem loop. So they mutated it back to the wild-type bacteriophage sequence, thinking that if the coat protein will bound less-tightly to the stem-loops, it will give a chance to Xrn1, the exonuclease, to degrade the MBS. She also increased the distance between loops from 30 to 50 nucleotides.

The results are incredible – there is no accumulation of aggergates, no co-localization to P-bodies(**), even in stress (at least for non-stress related mRNAs). The coding seqeunce and the 3’UTR (with the MBSv6) degrade at the same rate. And she could follow the life of mRNAs in live cells.

Seriously, go read the paper – it is excellent work.

* – I wish I had the time to do a better job with that response (e.g. do co-FISH), but lack of time  + microscope problems we had at the time led me to wrap up the thing quickly (well, as quickly as publishing works…).

** – There have been several interesting papers on P-bodies and stress granules lately – from Roy Parker, Karsten Weis, Jeff Chao and a few others. There are some contradictory results as to the roles of P-bodies in mRNA decay.  So I plan to write a long post about it (assuming I’ll find the time).

 

Tutucci E., Vera M., Biswas J., Garcia J., Parker R., and Singer RH. (2017) An improved MS2 system for accurate reporting of the mRNA life cycle. Nature Methods Epub Nov. 13 2017 doi:10.1038/nmeth.4502

Edit: I failed to notice that this was equal contribution of Evelina & Maria. My apologies to Maria. I now corrected the post to include both.

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