Keeping up with all the advances in science can be overwhelming. Sometimes, little things like acronyms can help people remember. When FMI Junior group leader Jeff Chao and his collaborators had developed a method for imaging the first round of translation in living cells and animals in 2015, they called it TRICK: Translating RNA Imaging by Coat protein Knock-off. Now comes the second delivery – and this time they decided to call it: TREAT, of course. With the new method the researchers are able to measure mRNA degradation, in single cells again, which nicely complements the TRICK method they developed earlier. With this a complete accounting of an individual transcript’s life from birth to death is now possible.
As reported in Molecular Cell, Chao’s group developed a fluorescent biosensor that allows the distinction of intact transcripts and degradation intermediates to study the dynamic life of mRNA within a cell. With the help of this sophisticated fluorescent microscopy method they are now able to capture the complex spatial and temporal dynamics of the degradation of single mRNA molecules in living cells. This is of special interest, as degradation of mRNA might play a more important role in regulation processes than previously thought. As Jeff Chao points out, "It has become increasingly clear that the regulation of mRNA degradation, particularly during development or rapid environmental changes, can dramatically influence RNA levels." While many of the RNA degradation steps have been characterized, we have so far been missing a clear picture of when and where degradation happens.
In the last years the development of quantitative fluorescent microscopy techniques to image single molecules of RNA has allowed many aspects of the mRNA lifecycle to be directly observed in living cells. But the new method helps to bridge a gap that has eluded scientist’s efforts so far. Single-molecule fluorescent in situ hybridization (smFISH) has enabled some aspects of mRNA degradation to be characterized, but the loss of signal resulting directly from the process being studied has prevented this approach from being widely applied. For their new method, Chao and his colleagues took advantage of a viral RNA structure that forms a knot-like structure. This pseudo-knot prevents the degradation of mRNA by Xrn1, a 5'-3' exoribonuclease. "And with the help of a multicolored biosensor containing these viral pseudo-knots, we were able to distin- guish between intact mRNA transcripts and mRNA transcripts that are being degraded", Chao explains. To visualize degradation, the scientists engineered a transcript that is labeled with two RNA-binding proteins fused to two distinct fluorescent tags: one of the proteins – PP7 (tagged with green fluorescent protein) – binds to the coding region of the mRNA, while the other – MS2 (with a red tag) – binds to the 3' untranslated region. Between PP7 and MS2, the scientists introduced the viral pseudo-knots. Thus labeled, the individual untranslated mRNAs appear yellow. As the RNA is degraded by XRN1, the green-tagged PP7 is displaced. However, at the position of the pseudo-knot, XRN1 degradation halts, which allows the detection of a quantifiable color change from yellow to red. And thus the scientists called this technique TREAT for 3(Three)'-RNA End Accumulation during Turnover.
The researchers describe their methods to establish this system in mammalian cell lines and Drosophila melanogaster oocytes, but they believe the principles can be applied to any experimental system. The method already led to interesting insights: The group has found that individual degradation events occur independently in the cytosol and that the degraded mRNAs did not accumulate in processing bodies. This is important because the processing bodies, membrane-less compartments that form during phase tra sitions, were thought to play a direct role in RNA degradation.
By Roland Fischer