Tag Archives: Singer lab

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

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.

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

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

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

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