I co-authored a Correspondence pre-print article that puts into question the Smartflare technology. SmartFlares (the commercial name of NanoFlares) are gold nanoparticles covered in oligos specific to a certain mRNA of interest (aka spherical nucleic acids). Supposedly, cells internalize these particles and, once the mRNA hybridize to the oligo, a complementary fluorecently labeled oligo is being unquenched and “flares”, indicating the presence of said mRNA. In this post I want to briefly mention the main topics of our pre-print, and expand on some points. I encourage readers to comment here or on Pubpeer on our article.
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. Continue reading
Posted in FISH, Gene expression, Journal club, MS2-like systems, stress response
Tagged mRNA decay, mRNA localization, MS2, quantitative microscopy, Singer lab, single molecule, yeast
My paper was recently published. I suggest that you read it before reading this post (it is an open access paper). In this paper we show that full-length mRNA molecules can be transferred between mammalian cells through membrane nanotube-like extensions that connect the cells.
Posted in Cell-Cell communication, epi, FISH, Gene expression, membranes, MS2-like systems, Transport & Trafficking
Tagged HHMI Janelia, Mammalian cell, membrane nanotubes, mRNA localization, MS2, my pics, personal experience, Singer lab, single molecule
Previously, on the story of MS2 in yeast: Last year, Roy Parker published a short article, in which he claimed that using the MS2 system in yeast causes the accumulation of 3′ RNA fragments, probably due to inhibition of mRNA degradation by the 5′ to 3′ exoribonuclease Xrn1. He argued that these findings put in question all the work on mRNA localization in yeast using the MS2 system. About a year later, we wrote a response to that article. We argued that, yes, such fragments exist, but 1. most of it stems from over-expression of the labeled mRNA. Parker agreed with that. 2. That these fragments accumulate in P-bodies, and are distinguishable from single mRNAs and we can discard cells which show these structures. 3. We argued that this might not be the case for every mRNA and should be tested on a case by case basis. 4. We and Parker agreed that the best way to determine if such fragments exist is by performing single-molecule FISH (smFISH) with double labeling – a set of probes for the length of the mRNA and a set of probes for the MS2 stem-loops. Now, a new paper from Karsten Weis’ lab shows more evidence, by doing smFISH, for the existence of these fragments.
I ended Part 1 after the morning session on pushing the boundaries of imaging.
After the amazing talks on imaging, I browsed the halls, visited some exhibitors, sampled a couple of exhibitor tech-talks. I later went to a mycrosymposium (#2: signaling in health & disease). This was mainly to see how this ePoster thing works, but also I promised Qunxiang Ong – with whom I discussed optogenetics the day before – to be at his presentation. He used a light-induced dimerization of signaling proteins to study the effect on neurite growth. The nice thing in his system was that the cells were plated in wells which were partly dark – so light-induction cannot take place in these regions. This allowed for analysis of neurite growth in lit vs “light-protected” regions of the same cell.
After this session, I attended my first “discussion table”. Continue reading
Posted in conferences & courses, epi, FISH, Gene expression, MS2-like systems, Optogenetics, Organelles, stress response, Transport & Trafficking, virology
Tagged ASAPbio, ascb15, bioRxiv, Mammalian cell, mRNA export, mRNA localization, PP7, QCBNet, quantitative microscopy, single molecule, yeast
The title of this post is also the title of a review paper that I co-authored with Adina Buxbaum, a recently graduated PhD student from Rob Singer’s lab. The review was published last week in Nature Reviews Molecular Cell biology.
In this paper we review some of the old and new methods to visualize mRNA. These include mostly FISH and MS2-like systems, which I’ve discussed extensively in this blog. There is also a short section (“box”) on quantitative analysis tools for mRNA localization imaging.
We then discuss the current knowledge on the mechanisms of mRNA localization and how it relates to the biology in two very distinct model systems – unicellular organisms (budding yeast) and the extremely polarized neuronal cell. We also discuss examples in other organisms from bacteria through fly to frog and mammals.
I’m biased, of course, but I think this turned out to be a balanced, comprehensive, yet not too detailed review paper that will benefit both beginners which are unfamiliar with the RNA localization field, as well as experts which are used to a single method or a single model organism.
Buxbaum, A., Haimovich, G., & Singer, R. (2014). In the right place at the right time: visualizing and understanding mRNA localization Nature Reviews Molecular Cell Biology DOI: 10.1038/nrm3918
Posted in adhesion & cell mobility, cytoskeleton, FISH, Gene expression, Image analysis, Journal club, MS2-like systems, Organelles, Transport & Trafficking
Tagged Bacteria, Fly, mRNA localization, neurons, quantitative microscopy, Singer lab, single molecule, yeast
How do you determine the localization of a single mRNA molecule in a living cell? Better yet – can you bring that mRNA where you want it?
I have briefly mentioned the MS2 system a while back. Essentially, The MS2 system to determine mRNA localization is composed of two parts: an RNA stem loop (referred to as MS2-coat protein (MCP) binding site, MBS) and the MCP – the coat protein from bacteriophage MS2.
MCP specifically binds the MBS (as a dimer).
A similar system with bacteriophage PP7 coat protein (PCP) and PBS was developed in our lab.
How does the MS2 system help to visualize mRNAs? Continue reading
Posted in adhesion & cell mobility, FCS, Journal club, Methods, MS2-like systems
Tagged Mammalian cell, mRNA localization, MS2, PP7, quantitative microscopy, Singer lab, single molecule, tethering