Category Archives: FISH

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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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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Imaging with CRISPR/Cas9

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.

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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

ASCB15 part 1

The ASCB meeting brings scientists from all levels to talk about cell biology, which is actually almost anything “biology”. But there’s also a full program dedicated to other matters, like science careers, science publishing, science communications and science policy. This is also a great venue for companies to show their products, and for organizations/institutions to recruit new members. If I remember the numbers correctly, there were over 550 oral presentations and over 2,700 posters. I overheard someone saying there were ~6000 people attending the meeting. I typically go to RNA meetings that are mostly in the lower 100’s of participants. So, to me, that’s a large meeting.

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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

sequencing localized RNA in single cells by FISH

To celebrate the 2-year anniversary of this blog, lets talk about the new Science paper in which the authors claim to performs in situ single cell, single molecule  RNA sequencing. So what’s the big deal? Well, RNA sequencing (RNA-Seq) has become a very common method to study gene expression. In many cases, RNA-Seq uses cell extraction from an entire cell population -thus averaging the RNA content of each individual cells. In recent years, single-cell RNA-Seq is becoming more feasible (for example). In this case, cells are sorted via flow cytometry so that one can sort individual cells into designated wells in a multi-well plate. Thus, RNA from a single cell can be sequenced. Though this process is becoming both efficient and accurate, you loose information about the cellular localization of the RNA. FISH is a method that enables to determine the accurate localization of the target mRNA. However, FISH is limited to only a small number of mRNAs. Using color barcoding of the FISH probes can increase the complexity that is achieved (i.e. – one can simultaneously detect multiple types of RNAs) but these are still only a few compared to the entire transcriptome. This new paper in Science combines FISH with RNA sequencing to give Fluorescent in situ sequencing (FISSEQ). How did they do that? First, they generated cDNA by performing reverse-transcription in fixed cells with random primers. The cDNA was then circularized and then amplified. One of the nucleotides has a reactive group so that the cDNA is cross-linked to the surrounding macromolecules. Thus, the cDNA is localized to the same location as the RNA.  The primer has an adapter sequence that can be used as template for sequencing or for FISH. They show some 3D FISH images of cells and tissues using a probe for this adapter. Pretty pictures, but not much info there. From here on, it gets trickier. For the sequencing, they used the SOLiD method. One problem was to reduce the spot density so that it will be sufficiently low to distinguish single molecules. If I understand them correctly, they modified the SOLiD sequencing probes so that the sequencing primers have mismatches that reduce the efficiency. The second problem was to identify auto fluorescence and other fluorescent artifacts in their images. Rather than fine-tuning a specific detection threshold, they relied on the fact that they are monitoring sequences. That means that the fluorescence at each spot should account for a known sequence (i.e. the colors pattern should change based on the sequence), whereas auto fluorescence should remain stable. So, they actually used no threshold at all.

The sequencing reaction cycles and images of the first 15 cycles in primary fibroblasts. Source: Lee JH et. al. (2014) Science 343:1360.

The sequencing reaction cycles and images of the first 15 cycles in primary fibroblasts. Source: Lee JH et. al. (2014) Science 343:1360.

They them showed a few applications of this method. One interesting feature was to show that the nucleus is enriched for non-coding and antisense RNAs compared to the cytoplasm that is enriched with mRNAs. Unfortunately, the nuclear/cytoplasm dichotomy was the only “localization” aspect in their paper. It would have been much more interesting to show mitochondrially-localized vs ER localized vs plasma membrane localized etc… Or, look at highly polarized cells like neurons and use FISSEQ to look at somatic vs dendritic or axonic RNAs. Another experiment they did was to look at the gene expression changes in response to a wound healing model. They found the expected increase in genes related to cell migration, and with some genes differentially expressed only in the migrating cells (at the “wound”) compared to contact inhibited cells that are close to them.  Again, It would have been even better if they could show the subcellular of these mRNAs – are they at focal adhesion sites or other unique sites in the migrating cells? Their results on the whole seem convincing, but I didn’t check all the little technical details.  One thing which is missing is a high resolution image showing the sequencing of just one RNA, compared to an auto fluorescence spot. All their images show one or many cells with multiple spots in different colors – but that is it. The authors say upfront that this is just a demonstration of their method, and that they expect it to improve in coming years, just like what happened with next-generation sequencing. We’ll see… ResearchBlogging.orgLee JH, Daugharthy ER, Scheiman J, Kalhor R, Yang JL, Ferrante TC, Terry R, Jeanty SS, Li C, Amamoto R, Peters DT, Turczyk BM, Marblestone AH, Inverso SA, Bernard A, Mali P, Rios X, Aach J, & Church GM (2014). Highly multiplexed subcellular RNA sequencing in situ. Science (New York, N.Y.), 343 (6177), 1360-3 PMID: 24578530   [post-pub note: this post was also published at the RNA-Seq blog, here].

Feb 2015 – the detailed protocol was published at Nature protocols.