Gene expression is just part of the story. After transcription, mRNA embarks on a journey out of the nucleus, undergoing many processes along the way, to the cytoplasm where translation can take place. Not all mRNA that is synthesised gets translated into protein, quite a lot of mRNA don’t make it. Instead they get degraded. The mRNA present in the cytoplasm is thus a balance between the levels of transcription vs. degradation. With such a dynamic process a dynamic visualisation system is required to study the kinetics and localisation of the events. Publishing in Molecular Cell, Horvathova et. al (1) have development a novel way of tracking the events of mRNA turnover.
A nifty trick
To study mRNA turnover, Horvathova’s team constructed a biosensor that they termed TREAT, exploiting elements from previous designs.
As can be seen from Figure 1 the biosensor consists of three key parts;
- PP7 bacteriophage binding loops
- A pseudoknot (PK)
- MS2 bacteriophage binding loops
The role of the two sets of binding loops is to allow binding of the proteins PP7 and MS2 binding protein (MCP) respectively. Both PP7 and MS2 are fused to fluorescent proteins which allows detection of the mRNA when the binding loops are accessible. Different fluorescent proteins, fluorescing at different wavelengths, must be fused to PP7 and MS2 for the biosensor to function as a sensor of mRNA turnover. In this case, PP7 was fused to a green fluorescent protein (GFP) whilst MCP was fused to Halo (a magenta fluorophore).
Pseudoknots are simply structured regions of RNA that form through the intercalation of stem loops. The pseudoknot used in this biosensor is from a flavivirus and prevents the action of a 5’-3’ exonuclease (a protein that degrades RNA starting at the 5’ end), known as Xrn1. Blockage of the nuclease is achieved through the sequestering of the 5’ phosphate group in the pseudoknot.
In this way, when the mRNA gets degraded by Xrn1, the PP7 binding loops are lost whilst the MS2 stay intact due to its protection from the pseudoknot. Thus, intact mRNA can be detected via the co-localisation of green and magenta fluorescence distinguishing it from partially degraded mRNA that keeps its magenta fluorescence (Figure 2).
Horvathova’s group thus developed a (three)’ –RNA end accumulation during turnover construct, a.k.a TREAT, an acronym that many hours, days or even weeks must have been spent to come up with.
Before using their construct to take live recordings they determined the half-life of TREAT. Expression of TREAT was induced for 2 hours and cells were subsequently fixed at successive time points and imaged to determine the states of the mRNA. These initial results showed that the degradation rate of intact TREAT was 1.63 ± 0.24 hr and 4.39 ± 0.47 hr for the stabilized 3’end TREAT.
Slicing in action
Live cell images usually last between 5-10 seconds, quite a bit shorter than the TREAT half live. Thus, to get sufficient data to record mRNA degradation, TREAT needed to be modified to increase its decay rate.
One pathway through which mRNAs are degraded is via short interfering RNAs (siRNAs). siRNAs recognise specific regions of mRNA via complementary base pairing (A with U and G with C) as a complex with a protein called Ago2. Ago2 cuts the mRNA (it ‘slices’ it) where siRNA has bound, triggering the mRNA for further degradation. Therefore, by inserting a siRNA binding site between the PP7 binding loops and the pseudoknot, TREAT can be more efficiently targeted for degradation. This addition knocked the half-life down to a mere ~10 minutes.
To image slicing in action, the authors rationalised that it could be visualised either by the separation of the green and magenta fluorescence or via the loss of the green signal. Amazingly, they observed the separation of the fluorophores (Figure 3) as they were cleaved apart by Ago2. The fact that they could see the green fluorescence suggests that siRNA-Ago2 mediated cleavage and degradation of cleaved mRNA are not that tightly coupled.
Imaging mRNA turnover, now that’s definitely a TREAT!
- Horvathova et al. The Dynamics of mRNA Turnover Revealed by Single-Molecule Imaging in Single Cells. Molecular Cell (2017) – read for even more experiments and videos they captured!