Single molecule FISH is currently the best method to get accurate measurements of mRNA levels at single molecule, single cell level in cell culture or tissue slices with a spatial resolution of ~200 nanometer (or less). One of the drawbacks of this method is the deterioration of the fluorescent signal (bleaching) of the organic dyes that are used to label the probes. Andrew Smith’s lab from University of Illinois now show how FISH can work with quantum dots instead of organic dyes. This provides better fluorophore stability and also the possibility to have more colors with less overlap of the emission spectra.
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
About a year and a half ago I wrote here about new uses of CRISPR/Cas9 as an imaging tool. In particular, I was excited about the possibility to use enzyme-dead Cas9 (dCas9) as an RNA binding protein for live imaging of mRNA. Unfortunately, in my hands this did not work (the dCas9 has exited the nucleus with non-targeting guide RNA at the same rate as with the specific guide RNA).
Last week, a new CRISPR tool was published in Nature, from Feng Zhang’s lab.
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.
Roy Parker recently sent a “Letter to the Editor“, published in RNA journal, in which he suggested that the MS2 system might not be best suited for live imaging of mRNA in budding yeast. According to Parker, the MS2 system inhibits the function of Xrn1, the major cytoplasmic 5′ to 3′ RNA exonuclease in budding yeast, causing us to image mostly the remaining 3’UTR fragments. Thus, he claims, it is possible that interpertation of mRNA localization data using this system in yeast can be faulty. We wrote a response to his letter which just opened the debate even further.
But lets start with his Letter:
Posted in FISH, Gene expression, Journal club, MS2-like systems
Tagged FISH-Quant, mRNA decay, MS2, my pics, personal experience, quantitative microscopy, Singer lab, yeast
The hottest buzz-word in biology today is CRISPR: an adaptive immune system in bacteria and archea. At its basis is a nuclease, named Cas9, which is targeted to DNA by a short single-guide RNA (sgRNA). This turned out to be a very useful system for genome engineering in any organism due to its specificity (provided by the sgRNA) and its simplicity (all you need is to express the Cas9 and sgRNA in the cell). However, this system can also be used for other purposes. One such use is modulation of gene expression, for example by targeting a nuclease dead Cas9 (dCas9) fused to a transcription activator or repressor to promoter regions. Another such use is for imaging.
Here, I’ll described how Cas9 can be used to visualize specific DNA loci or specific RNA transcripts in fixed and live cells.
Posted in CRISPR/Cas9, FISH, Gene expression, Genetics, Journal club, Whole tissue imaging
Tagged CASFISH, CRISPR, DNA FISH, HaloTag, Mammalian cell, quantitative microscopy, RCas9, Singer lab, stress granules