Tag Archives: PP7

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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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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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

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.

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When two halves equal zero (background)

Fluorescent imaging is all about the contrast between the signal and the background. For imaging to be successful, the signal should be clear above the background. Background fluorescence can come from free/non-specific fluorescent probe, autofluorescence, and out of focus fluorescence.

There are two major strategies to improve signal/background ratio.

The first is to increase the signal. We do that by choosing brighter fluorescent molecules, by increasing the number of fluorescent probes per target, by using more than one color per target, by having photoactivatable probes etc…

The second strategy is to reduce the background. The wash step in IF and FISH protocols is intended to remove excess, non-specific bound, probe. There are even more extensive wash protocols.  We have many type of microscopes that are designed to reduce out-of-focus light (these include confocal, TIRF, multi-photon, and SPIM).  In yeast imaging, we sometimes add an excess of adenine to the culture media, since many strains are defective in adenine biosynthesis, and accumulate a red intermediate molecule. In the field of single molecule live mRNA imaging, we usually add a nuclear localization signal (NLS) to the fluorescently tagged RNA binding protein, in order to reduce its cytoplasmic fluorescence.

Now, my lab-mate Bin Wu develop a system that he calls “Background free imaging of single mRNAs in live cells using split fluorescent proteins”.

The idea is to combine the two most common systems – the MS2 and the PP7 systems, so that the MS2 binding sequence (MBS) and PP7 binding sequence (PBS) will be in tandem. Then the MS2 coat protein (MCP) will be fused to one half of a fluorescent protein (Venus) and PCP will be tagged with the other half. Only when MCP-VenusN and PCP-VenusC are in very close proximity (e.g. bound to the MBS and PBS, respectively) the two halves can bind to form Venus, which fluoresce in bright green-yellow.  Add 12 of these tandem repeats to the mRNA and you have 24 fluorescent proteins on the mRNA in the cytoplasm, with, theoretically, zero unbound fluorescent protein in the cytoplasm, hence “zero background”.

The system has some limitations. For one thing, the protein levels must be low, since the fluorescent protein halves can self-associate at high concentrations independent of interaction with the mRNA. Also, since it takes the fluorescent protein some time to mature, it is not useful to study short-lived mRNAs, or  transcription in live cells, since by the time it matures, the mRNA has already left the nucleus.
ResearchBlogging.orgWu B, Chen J, & Singer RH (2014). Background free imaging of single mRNAs in live cells using split fluorescent proteins. Scientific reports, 4 PMID: 24402470

Two colors, single mRNA, and a multitude of transcription rates

Imaging single mRNA molecules in live cells has proved to be very useful in studying mRNA localization, as well as mRNA transcription. Specifically, by using the MS2-like systems, it is possible to follow the synthesis of single mRNAs, thus determining the rate of transcription.

Tagging an mRNA with MS2-like systems allows following  the transcription of that gene by imaging. Adapted from Larson et al. (2011)

Tagging an mRNA with MS2-like systems allows following the transcription of that gene by imaging. Adapted from Larson et al. (2011)

By putting the MBS (MS2-coat protein binding sequence) or PBS ((PP7-coat protein binding sequence) at the 5’UTR of a gene, one can follow the fluctuation of fluorescence at the transcription site. In essence, the MS2-tagged mRNA should appear as a bright spot in the nucleus as soon as the MBS sequence is transcribed. Once the mRNA is fully processed and exported from the nucleus, the fluorescence signal of the transcription site should decrease.

What happens if you have the same gene being transcribed simultaneously by multiple polymerases?

Well if the MBS is at the 3’UTR, then the duration of the MS2 signal at the transcription site for each mRNA should be short (the time it takes to complete synthesis of the 3’UTR and the 3’-end processing).

However, if the MBS is at the 5’UTR, then the signal lasts from early elongation until 3’-end processing and export. For the GLT1 gene in yeast, it is ~200 seconds. Since that gene is ~6400 bases long, than we can calculate that the rate of RNA polymerase is less than 32 bases per second (we do not know the 3’-end processing/termination time).

Time-dependent activity of individual reporter genes (PP7-GFP, green; Nup49-tomato, red). At time t = 0 min, both cells show a TS near the periphery of the nucleus. At t = 2 min, an additional TS corresponding to the duplicated gene has turned on in the upper cell (white and blue arrows). Right: Intensity trace (green line) of the transciption site. The gray line is the intensity of a cytosolic mRNA; the black line is a background intensity at an arbitrary position in the nucleus. Adapted from Larson et al (2011).

Time-dependent activity of individual reporter genes (PP7-GFP, green; Nup49-tomato, red). At time t = 0 min, both cells show a TS near the periphery of the nucleus. At t = 2 min, an additional TS corresponding to the duplicated gene has turned on in the upper cell (white and blue arrows). Right: Intensity trace (green line) of the transciption site. The gray line is the intensity of a cytosolic mRNA; the black line is a background intensity at an arbitrary position in the nucleus. Adapted from Larson et al (2011).

We can calculate the real elongation rate by subtracting the signal dwelling time (how long we see the fluorescent spot) with the 3’UTR MBS from the dwelling time with the 5’UTR. Using this method it was possible to calculate the elongation rate of RNA polymerase II on the MDN1 gene in yeast (it is the longest gene in yeast, almost 15,000 bases long). The rate was found to be 20±8 bases per second. Knowing that, we can subtract the time needed for the remaining of the 3’UTR downstream of the 3’UTR MBS, and what is left is the termination time. For the MDN1 gene it is 70 seconds (i.e. it takes the 3’-end processing and export machinery more than a minute to get the mRNA away from the nucleus).

However, this method requires measuring different genes (one with 5’UTR-MBS and another with 3’UTR-MBS) in different cells, getting their average, then subtracting. It would be much more accurate to measure a single gene in a single cell and get an absolute value from that measurement.

Here comes the development of two-color system. Instead of using just MBS, we insert the MBS at the 3’UTR and the PBS  at the 5’UTR. We then express PCP-GFP and MCP-RFP and we can have a two colored mRNA. Upon expression, the GFP signal comes first and the RFP signal comes later. By measuring the duration from appearance of GFP to appearance of RFP, we can directly get the elongation rate of a single RNA polymerase enzyme, on a single gene, in a single cell.

That is exactly what Sami from our lab did. He tagged the MDN1 gene with the PBS and MBS and measured the elongation rate in single cells. On average, the rate was found to be 25 bases per second. This is agreeable with the results I mentioned above.

(a) Schematic showing the tagging of MDN1 gene wirh 5' PP7 loops and 3' MS2 loops. (b) Appearance of 5′ (magenta) and 3′ (green) transcription site signals at indicated times after induction. Final frames show an overlay of both signals. Adapted from Hocine et al. (2012)

(a) Schematic showing the tagging of MDN1 gene wirh 5′ PP7 loops and 3′ MS2 loops. (b) Appearance of 5′ (magenta) and 3′ (green) transcription site signals at indicated times after induction. Final frames show an overlay of both signals. Adapted from Hocine et al. (2012)

But more interesting was the high variability between cells. The elongation rates of the same gene in differnt cells ranged from as slow as 14 bases per second to the fast rate of 61 bases per second.

What does it mean? That the text-book view that transcription elongation is constant is not true. In recent years it was shown by different methods that transcription elongation is regulated, and that RNA polymerase II is prone to pausing. Here is direct evidence that indeed the same gene is elongated at different rates in different cells. Which means that we still have a lot to learn about transcription in general, and elongation in particular.

ResearchBlogging.orgLarson DR, Zenklusen D, Wu B, Chao JA, & Singer RH (2011). Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science (New York, N.Y.), 332 (6028), 475-8 PMID: 21512033
Hocine S, Raymond P, Zenklusen D, Chao JA, & Singer RH (2012). Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nature methods PMID: 23263691

Put that mRNA where you want it

How do you determine the localization of a single mRNA molecule in a living cell? Better yet – can you bring that mRNA where you want it?

I have briefly mentioned the MS2 system a while back. Essentially, The MS2 system to determine mRNA localization is composed of two parts: an RNA stem loop (referred to as MS2-coat protein (MCP) binding site, MBS) and the MCP – the coat protein from bacteriophage MS2.

MCP specifically binds the MBS (as a dimer).

A similar system with bacteriophage PP7 coat protein (PCP) and PBS was developed in our lab.

How does the MS2 system help to visualize mRNAs? Continue reading