Tag Archives: quantitative microscopy

New CRIPSR-based RNA imaging tool

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. Briefly, Zhang’s team found in the past a new type of Cas protein which was named C2c2 and later Cas13. Unlike Cas9, which typically binds and cuts DNA, Cas13 specifically binds and cuts RNA. In this paper, they isolated a Cas13a variant from Leptotrichia Wadei (LwaCas13a),  and used it as a programmable tool to knock-down expression. As a tool for knowckdown it is pretty awesome – it is comparable to shRNA in terms of knock-down (~40-90%), but it has three big advantages:

1. Unlike shRNA, LwaCas13a can also target nuclear RNA.

2. It can be used easily for multiplexed knock-down of sereval RNAs.

3. Amazingly, it seems that it has NO OFF-TARGET effect. They used RNA-seq to look at the change in expression level of all RNAs in the cells. Whereas shRNA showed hundreds of off-target knocked-down RNAs, LwaCas13a was very specific and only the targeted mRNA was knocked-down.

They then mutated LwaCas13a to create a dead variant (dLwaCas13a) and asked if it can be used for pull-down assays and for live imaging.

Just like the RNA-dCas9, the pull down efficiency of beta-actin mRNA wasn’t great (2-3 fold over non-targeting guide RNA). However, for Luciferase mRNA, this ratio was better (8-11 fold enrichment). So, this has good potential. Too bad they did not compare it to puul down using MS2-labeling and see if the same proteins are pulled down with the mRNA.

And now I come to my problem with this paper – the imaging.

Just like the RNA-Cas9 paper, they imaged beta-actin mRNA with a single guide RNA and single GFP fused to dLwaCas13a. dLwaCas13a was nuclear localized, but exited the nucleus when specific guide RNA was used. However, they claim only a 3-3.7 fold enrichment of cytoplasmic/nuclear ratio compared to non-targeting guide RNA. They acknowledge that there is neclaer escape due to nuclear off-target binding or other reasons. They added a negative feedback system to improve SNR. Still, the best they got was 3.7-fold. So, if you look at a specific cell – how can you tell if you are looking on RNA-bound or not? you can’t.

They also compared their labeling to FISH staining. Their FISH looks much better than in the RNA-Cas9 paper, really single spots. But to say that it co-localizes? There is more-or less uniform cytoplasmic green staining. Of course it will “co-localize” with the cytoplasmic beta-actin mRNA FISH spots. At the very least they should have done a control with FISH against another highly expressed mRNA (e.g. GAPDH), or perform beta-actin FISH of cells expressing a guide RNA against a differnt mRNA, and not just compare to the non-targeting control.

Last point – stress granules again? I guess it’s just an easy assay to do, that also gets the RNA in a concentrated blob: good if you don’t have single molecule resolution. But nobody really knows what SGs are and only now starting to figure out how and why they form.

They claim that when they induce stress granules (SG) formation, the GFP-dLwaCas13a co-localizes with the SG marker. However, their images are not very good. You see something in Fig 4 (only 2 cells in the control!) but Extended Fig 10 is terrible. Low resolution, small images – can anyone tell from this figure if there is or isnt co-localization, and get some qualitative, if not quantitative sense?

They do provide quantitative data, but although it could be statistically significant, it does not look striking. Really marginal differnece compared to the non-targeting guide RNA (or non-stressed cells).

Is this a good tool for RNA imaging? I don’t think so. Not until you can get single-molecule resolution.

 

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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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Counting exosome secretion

Last month I wrote a post about exosome internalization by recipient cells.  One of the topics I discussed was the lack of good quantitative data in the exosomal field, and what the current data tells us about the efficiency and capacity of exosome-mediate cell-to-cell communiation.

Today I came across an interesting paper in which the researchers try to get quantitative data of exosome secretion by the donor cells.

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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.

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The wild ride of the exosomes

Exosomes are extracellular vesicles that are thought to mediate cell-to-cell communication in eukaryotes. Briefly, exosomes are 50-100 nanometer (nm) sized vesicles produced by the endosomal system. They are exported out of the cell and can be found in every bodily fluid: plasma, saliva, milk, urine and more. These vesicles then enter recipient cells, and the cargo they carry (proteins, RNA molecules and lipids) modulate the physiology and/or gene expression of the recipient cell. Exosomes catch a lot of attention lately because of their clinical significance. First, exosomes might be used as biomarkers for some diseases (most importantly tumors). Second, they are being considered for therapeutics as a delivery 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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