Tag Archives: yeast

MS2 mRNA imaging in yeast: more evidence for artefacts

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.

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Design guidlines for tandem fluorescent timers

Almost 4 years ago, I wrote a post on tandem fluorescent timers (tFTs). The idea is to have two different fluorescent proteins fused together to the protein of interest. In the paper from 4 years ago, it was superfolder GFP (sfGFP) and mCherry. sfGFP matures very fast (within minutes) and mCherry  matures more slowly (t1/2 ~40min). The ratio beween green to red fluorescent signal indicates the percentage of new vs old proteins, thus acts as a “timer”.  This latests paper on tFTs from the same group of Michael Knop’s lab, found that analyzing tFTs might be more complicated due to some possible problems of this system.

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Does bound MS2 coat protein inhibit mRNA decay?

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:

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ASCB15 – part 2

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

In the right place at the right time: visualizing and understanding mRNA localization

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.
ResearchBlogging.orgBuxbaum, 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

FISEB 2014 – day 2

Due to crappy Wi-Fi at hotel, this entry will be short. I’ll try to expand once I get back home.

Anyway, today was very interesting.

At the “early bird” session, I heard about CyTOF. Essentially, instead of using a few fluorescent markers for FACS sorting of different cell types, they offer conjugating the tagging antibodies with rare heavy metal isotopes. they claim that these are not found in cells, so the background should be zero. They have >30 different isotopes they can use, and the detection is by mass spectrometry – so very accurate and distinct identification.

Next was a session on gene expression. I won’t go into details, particularly since much is unpublished yet, but Tzachi Pilpel’s talk was amazing. Who knew tRNA may have anything to do with cancer research?

As per usual, Orna Amster-Choder talked about RNA localization in bacteria with lovely images and great data.

Jeff Gerst from Weizmann discovered a possible new mechanism of mRNA transport in yeast, using the MS2 system in very neat ways.

The next session, called “oral poster 1”  featured short talks. The most interesting to me were about mRNA methylation and about how the DNA sequence surrounding consensus sequence for DNA binding proteins affects this binding. some nice insights.

The last session I attended was about the effect of tumor microenvironment on tumor progression and treatments. Heard some amazing stories. Hope still exist to cure cancer…

Tomorrow is my lecture. Excitement!

FISEB 2014 meeting -day 1

FISEB meeting happens every three years, and it includes participants from 28 different experimental biology societies in Israel. It is the best meeting to learn about biological-medical research performed in Israel at all fields and doctrines.

4 days, 8-10 parallel sessions, hundreds of lectures, >1000 posters, >2200 participants.

The first day started by a plenary lecture by Aryeh Warshel, Nobel lauret. He is really far from my field, and his lecture was very much confusing to me. But he has nice cartoons 🙂 The bottom line – enzymes are able to catalyze reactions due to electrostatic connections that are maintained stable (unlike in water).

From the afternoon sessions, I chose “signaling pathways & networks”. Relevant to this blog:

Yoav Henis from Tel-Aviv Uni. talked about oligomerization of TGF-beta receptors. he used a method he calls “co-patching”, which is essentially IF with two different antibodies for two receptor subunits. homodimerization will yield single color “patch” whereas heterodimerization will yield an overlap of both colors (co-patch). He then looked at the % of co-patch with different receptor subunits with/without ligand, or with mutants.

Maya Schulinder from Weizmann Institute talked about the contacts between mitochondria and other organelles (ER, vacuole) in yeast. These contacts are important for lipid metabolism. She new about the mito-ER contact but found there must be a second contact (bypass mechanism). She used an interesting screen method to find the bypass mechanism to the mito-ER contact: she expressed one of the contact protein as a GFP fusion. She expected that if the bypass mechanism and the mito-ER contact “share the load” of lipid metabolism, then deletion of the bypass will increase the number of the mito-ER contacts to compensate. Using automation, she imaged 6200 deletion mutants (from the yeast deletion library) each expressing this GFP fusion. As expected, she found 4 candidates which turned out to be very interesting.

Roni Seger from Weizmann showed that targeting the nuclear localization signal of ERK can be a novel cure for certain pathologies, including certain types of cancer.

On the other hand, Maya Zigler from the Hebrew Uni. suggested another new idea to cure cancer – by inducing the surrounding immune cells to destroy the tumor.

Ido Amit from Weizmann as well told us that we may not really know all the different types of cells that exist. What most people do, particularly in immunology, is rely on one or two known “markers” and use FACS or other methods to sort the cells based on these markers. However, some of the markers overlap. and there may be cells for which we do not have any markers and they “disappear” in the crowd of unsorted cells. or, the could be further sub-types we do not know about. So he approached the problem in an unbiased way – he took all the cells in the spleen, and did single cell RNA seq to individual cells from the spleen. Thus, each cell type has several hundred/thousand “markers” based on gene expression profiles. Not only did this method agree with the common FACS sorting markers, but he identified several sub-types unknown before.  Expect his paper this month in Science. His paper just got published in Science.

Finally, Yaron Shav-tal from Bar-Ilan Uni. used the MS2 system to study how perturbing the signaling pathway of serum stimulation affects transcription of beta-actin gene. As per usual – very neat job and interesting results.