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.
The Singer group has used the FlAsH-ReAsH system, where a tetracysteine motif binds to a biarsenical dye, to detect newly synthesized peptides in fibroblasts, but have abandoned this approach because of the chemical toxicity to cells and the background of the dye. Moreover, This system still does not allow for single molecule resolution.
Fluorescent non-canonical amino acid tagging (FUNCAT) is system developed in Erin Schuman’s lab to visualize protein production in neurons. Though it is a fun system, it provides information on total protein production, not of a specific protein. Furthermore, the shortest incubation time with the tag was 10 minutes – fairly long for single molecule resolution. However, there are reports of even a 5 second pulse. I wasn’t convinced.
I’ve heard about TRICK when I joined the Singer lab three years ago. Translating RNA imaging by coat protein knock-off (TRICK) is a system developed by Jeff Chao (then a postdoc at the lab, now has his own lab in Basel, Switzerland). The idea is fairly simple – instead of trying to capture the protein being produced, let us examine the “translational state” of the mRNA.
TRICK takes advantage of both the MS2 and the PP7 systems. The MBS (MS2 cap protein binding sequence) tags the 3’UTR, and is therefore a stable tag on the mRNA. However, the PBS (PP7 cap protein binding sequence) was distributed in the coding sequence (usually just upstream to the stop codon). Expressing MCP-RFP and PCP-GFP yields a dualy-labeled mRNA. Once the mRNA is translated, the ribosome traverses the coding sequence and, upon translating the PBS, displaces the PCP-GFP bound to the stem-loops, yielding a red-only mRNA. Thus the loss of the GFP signal indicates that the mRNA was translated.
Indeed, most of the cytoplasmic mRNAs are tagged only by RFP, indicating that they have been translated at least once. Here also lies a disadvantage of this system – it only allows detection of the first round of translation, because once displaced, the PCP-GFP (which has a nuclear localization signal, NLS) quickly diffuses away from the mRNA and, presumably is imported back to the nucleus. [The NLS is required, in order to reduce cytoplasmic background].
To prove the point, Jeff shows that addition of translation inhibitors results in mostly dual-colored mRNAs.
Translation in the nucleus has been the subject of much debate (see also the paper I linked to above, re: FUNCAT in 5 seconds). The TRICK system allowed examining this issue, at least for a specific mRNA. Indeed, TRICK analysis did not suggest that any significant translation occurs in the nucleus, at least not of that specific mRNA and at least not the entire length of the coding sequence.
TRICK was further used to examine the distance by which mRNAs are being translated for the first time after nuclear export (most are translated fairly soon after export, though some mRNA molecules are being translated minutes after their export) as well as the translational state of the mRNAs upon stress and assembly into P-bodies.
Many mRNAs are being translated only at a specific location or at a specific time-point. For example, upon budding, the ASH1 mRNA in yeast is translationally repressed during its transport to the bud tip. OSKAR mRNA in translated only when it reaches the pole plasm area of a stage 10 oocyte. TRICK is a great system to detect where and when such mRNAs are first translated. So, in collaboration with Anne Ephrussi, they showed indeed that OSKAR-TRICK mRNA is not translated until the exact time in development.
The first round of translation is also the cell’s quality control mechanism, in particular for nonsense-mediated decay (NMD). Since OSKAR-TRICK did not go through translation until a late stage in oogenesis, it implies that only at that developmental stage OSKAR mRNA goes through NMD. The greater implication is that though for some mRNAs NMD may occur immediately upon export, translationally-repressed/ transported mRNAs may experience quality control only upon arrival at their destination or temporal activation.
TRICK can be used to ask many other questions, such as questions related to NMD and mRNA decay in general; questions about “non-coding RNAs”; Questions about IRES and other forms of alternative translation and many more.
I’m happy for Jeff that its finally published.
Halstead JM, Lionnet T, Wilbertz JH, Wippich F, Ephrussi A, Singer RH, & Chao JA (2015). Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals. Science (New York, N.Y.), 347 (6228), 1367-671 PMID: 25792328
Yu, J. (2006). Probing Gene Expression in Live Cells, One Protein Molecule at a Time Science, 311 (5767), 1600-1603 DOI: 10.1126/science.1119623
Dieterich DC, Hodas JJ, Gouzer G, Shadrin IY, Ngo JT, Triller A, Tirrell DA, & Schuman EM (2010). In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nature neuroscience, 13 (7), 897-905 PMID: 20543841
Rodriguez, A., Shenoy, S., Singer, R., & Condeelis, J. (2006). Visualization of mRNA translation in living cells The Journal of Cell Biology, 175 (1), 67-76 DOI: 10.1083/jcb.200512137
Baboo S, Bhushan B, Jiang H, Grovenor CR, Pierre P, Davis BG, & Cook PR (2014). Most human proteins made in both nucleus and cytoplasm turn over within minutes. PloS one, 9 (6) PMID: 24911415