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?

Well, the MCP can be fused to a fluorescent protein. The MBS can be fused to the mRNA of choice (usually at the 3’ un-translated region, UTR). If we add two stem loops, we get a dimer of MCP bound to the mRNA, and hence two GFPs bound to the mRNA. Usually, the MBS is multiplied (24x is good) so we can get (a maximum of) 48 GFP proteins bound to the mRNA.

It is necessary to have multiple MBS since not all MCP is bound to the mRNAs, thus creating a background fluorescence of free MCP-FP in the cytoplasm. In many cases a nuclear localization signal (NLS) is added to the MCP-FP to concentrate it in the nucleus and reduce cytoplasmic background.

A little detour to talk about the MCP occupancy on the mRNA:

A recent paper from our lab used FCS to measure MCP and PCP, dimerization and binding to mRNA. What was found was that MCP dimerizes at concentrations of >400nM, whereas PCP dimerizes at <20nM. This means that MCP levels in the cytoplasm should be high enough to allow dimerization, which leads to high background.

Based on this result, they developed a tandem MCP (tdMCP)-GFP or tdPCP-GFP fusion protein. Thus, the coat protein is already “dimerized” and does not require a high cytoplasmic concentration.

Using the tdPCP they showed that the brightness of an mRNA with 24xPBS is equal to about 24 GFPs, i.e. full capacity. For PCP-GFP the brightness was about 48 GFPs – again, full capacity.

However, for MCP and tdMCP only occupied ~ half the sites (26 and 13, respectively).

Back to localization of the mRNA

There have been many papers that used MS2-like systems to study mRNA localization in live cells – from yeast to mammals. Furthermore, our lab has developed a transgenic mouse with β-actin-MBS.

Using this mouse, we are able to follow β-actin localization in different cell types, conditions and mutants. A paper published by several lab members showed that β-actin mRNA is localized to focal adhesions. In fact, this localization is required for the directionality of movement of mouse embryonic fibroblasts (MEFs) (this was shown by knock-out of ZBP1, a protein known to be required for β-actin localization).

beta-actin mRNA localization using the MS2 system in MEFs. Sources: Katz et al. (2012) Gen Dev 26:1885

beta-actin mRNA localization using the MS2 system in live MEFs. Green – free GFP as cytoplasmic marker. Red – TagRTPt-labeled beta-actin mRNA Source: Katz et al. (2012) Gen Dev 26:1885

This was not easy to demonstrate. There are several thousand β-actin mRNAs in the cell and they constantly move. What Zack and the others developed was an algorithm to track mRNAs close to focal adhesions, and measure their dwell time – i.e. how long will an mRNA remain close to the focal adhesion. Dwelling is determined by the distance the mRNA is crossing, as well as its directionality. What they showed that mRNAs close to adhesion sites dwell there for several minutes (supposedly getting translated there).

Where should I put this mRNA?

β-actin mRNA has a zipcode sequence in its 3’UTR that directs the mRNA to the correct localization in the cell. Many localizaed mRNAs has such “zipcode” sequences.

But what would happen if the mRNA mis-localize?

One way to test it is to delete the zipcode, or replace it with a zipcode of another mRNA. But what if you wanted to bring it to a particular location but have no known zipcode for it?

Here comes again the power of the MS2 system.

What Zack did was to fuse the MCP to a known focal adhesion associated protein (vinculin). The Vinculin-tdMCP-GFP now not only binds the mRNA but also tether it to focal adhesions. The result was that focal adhesions were larger and more stable and could also partially save the ZBP1 K/O phenotype.

Tethering an mRNA to a specific location in the cell (focal adhesion in this case) using the MS2 system. Source: Katz et al. (2012) Gen Dev 26:1885

Tethering an mRNA to a specific location in the cell (focal adhesion in this case) using the MS2 system. Source: Katz et al. (2012) Gen Dev 26:1885

This novel approach of tethering mRNA to specific locations in the cell will be very helpful in understanding local translation of mRNAs, and how it affects cell dynamics, structures, viability, differentiation and more.

ResearchBlogging.org
Katz ZB, Wells AL, Park HY, Wu B, Shenoy SM, & Singer RH (2012). β-Actin mRNA compartmentalization enhances focal adhesion stability and directs cell migration. Genes & development, 26 (17), 1885-90 PMID: 22948660
Wu B, Chao JA, & Singer RH (2012). Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophysical journal, 102 (12), 2936-44 PMID: 22735544
Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, & Singer RH (2011). A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nature methods, 8 (2), 165-70 PMID: 21240280

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