Tag Archives: MS2

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.

Continue reading

Advertisements

Imaging translation of single mRNAs in live cells

Translating the information encoded in mRNAs into proteins is one of the most basic processes in biology. The mechanism requires a machinery (i.e. ribosomes) and components (mRNA template, charged tRNAs, regulatory factors, energy) that are shared by all organisms on Earth. We’ve learned a great deal about translation over the last century. We know how it works, how it is being regulated at many levels and under varuious conditions. We know the structures of the components. We have drugs that can inhibit translation. With the emergance of next-gen sequencing, we can now perform ribosome profiling and determine exatly which mRNAs are being translated, how many ribosomes occupay each mRNA species and where these ribosomes “sit” on the mRNA, on average. New biochemical approaches like SILAC and PUNCH-P can quantifiy newly synthesized proteins & peptides. Yet, all of that information comes from population studies, typically whole cell populations. Rarely, whole transcriptome/ribosome analysis of a single cell is performed. Still, there is no dynamic information of translation, since cells are fixed and/or lysed. And there is no spatial information regarding where in the cell translation occurs (poor spatial information can be determined if cell fractionation is performed, which is never a perfect separation of organelles/regions and we are still not at the stage of single organelle sequencing).

Imaging translation in single cells is intended to provide both spatial and dynamic information on translation at the single cell and, hopefully, single mRNA molecule resolution. Recently, four papers were published (on the same day!) providing information on translation of single mRNAs. Here is a summary of these papers.

Continue reading

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:

Continue reading

Visualizing translation: insert TRICK pun here

Unlike transcription, it is much harder to image translation at the single molecule level. The reasons are numerous. For starters, transcription sites (TS) are fairly immobile, whereas mRNAs, ribosomes and proteins move freely in the cytoplasm, often very fast. Then there are only a few TS per nucleus, but multiple mRNAs are translating in the cytoplasm. Next, there’s the issue of signal to noise – at the transcription site, the cell often produces multiple RNAs, thus any tagging on the RNA is amplified at the transcription site.  Last, it is fairly easy to detect the transcription product – RNA – at a single-molecule resolution due to multiple tagging on a single molecule (either by FISH or MS2-like systems). However, it is much more difficult to detect a single protein, be it by fluorescent protein tagging, or other ways (e.g. FabLEMs).

The rate of translation is ~5 amino acids  per second, less than 4 minutes to a protein 1000 amino-acids long. This is faster than the folding and maturation rate of most of even the fastest-folding fluorescent proteins. This means that by the time the protein fluoresce, it already left the ribosome. However, attempts were made in the past with some success.

Continue reading

Looking at single mRNAs in neurons hints at memory formation

It is postulated that learning and memory are modulated by synaptic plasticity – molecular changes  that result in changes in the synapse morphology and signaling capacity. Local protein translation is considered important for synaptic plasticity. Two works from our lab were published last month (back to back!) in Science. Both papers deal with how beta-actin mRNA localization and dynamics in neurons may account for local protein translation upon stimulation, and hence, may supply insight into memory formation.

The first paper by Adina Buxbaum shows that beta-actin mRNAs in dendrites are “unmasked” upon activation of the dendrites. Using single molecule FISH, She noticed that the average number of probes bound to the mRNAs in dendrites (but not in adjacent glia cells) was lower than expects, and this number increased upon stimulation. Not only that, there were more mRNAs in the stimulated dendrites. This indicated masking by a protein “coating” that prevented FISH probe binding in the unstimulated cells. A modified FISH protocol which included a protease digestion step prior to probe hybridization showed that indeed the mRNAs were masked by proteins.

single molecule FISH for beta-actin mRNA in dendrites shows that mRNAs in unstimulated neurons are masked. A) Unstimulated neuron. B) stimulated neuron showing increased number of spots. C) Unstimulated neuron, in which the fixed cells were digested with protease prior to FISH probe hybridization. Source: Buxbaum, Wu & Singer (2014). Science Vol. 343  pp. 419-422

single molecule FISH for beta-actin mRNA in dendrites shows that mRNAs in unstimulated neurons are masked. A) Unstimulated neuron. B) stimulated neuron showing increased number of spots. C) Unstimulated neuron, in which the fixed cells were digested with protease prior to FISH probe hybridization. Source: Buxbaum, Wu & Singer (2014). Science Vol. 343 pp. 419-422

 

She further showed that this masking relates to other mRNAs, as well as to ribosomes, and that this is due to a metabolic process resulting from stimulation. Thus, this unmasking process may be a way to “activate” localized mRNAs for translation.

Apart from being a very neat paper technically and biologically, I think it was exceptionally entertaining to begin her paper by quoting an 1894 work by Cajal, the father of neuroscience.

The second paper by Hye-Yoon Park follows the dynamics of single molecule endogenous beta-actin mRNAs in neurons by live imaging, using the MS2 system. She shows movement of mRNAs along dendrites, as well as some events of merging or splitting – suggesting that some mRNAs are packed together in larger granules – which may regulate local translation. She also looked at brain slices, visualizing beta actin transcription dynamics. This is an important achievement since it is much harder to look at mRNA dynamics in tissue slices than in single cells on plate, due to background fluorescence. Though some biological insight is derived here, this is more of a “new technology” report.

Live imaging of beta-actin mRNAs in dendrites (movie. Source: Park HY et al. (2014) Science Vol. 343 pp. 422-424)

These papers are just the beginning of a long-term story of how mRNA localization and local translation are regulated in neurons.  A lot of cool experiments are being done in our lab in this regard and I’ll report more as they are published.

ResearchBlogging.orgBuxbaum AR, Wu B, & Singer RH (2014). Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science (New York, N.Y.), 343 (6169), 419-22 PMID: 24458642
Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, & Singer RH (2014). Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science (New York, N.Y.), 343 (6169), 422-4 PMID: 24458643

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.