Real-time quantification of single RNA translation dynamics in living cells
Assistant Professor Timothy Stasevich, Colorado State University, USA
Professor Stasevich has developed technology to image single RNA translation dynamics in living cells. Using high-affinity antibody-based probes, multimerized epitope tags, and single molecule microscopy, we are able to visualize and quantify the emergence of nascent protein chains from single pre-marked RNA1. In this talk, he will describe this technology as well as a new multi-frame tag that extends the technology to enable real-time quantification of single RNA translation kinetics in any two of the three possible open reading frames. As a first application of the multi-frame tag, he uses it to dissect the kinetics of the HIV-1 frameshift sequence. Whereas previous bulk assays have shown this sequence leads to ~10% frameshifted product, it was not clear if all RNA frameshift with ~10% efficiency or if instead ~10% of RNA frameshift with ~100% efficiency. Interestingly, our live-cell data suggest the latter scenario, where a small subset of genetically identical RNA frameshift with high efficiency. The origin of this heterogeneity is not yet clear, but experiments are beginning to implicate a ribosomal pause that leads to a higher than normal density of ribosomes near the frameshift sequence on frameshifting RNA.
Imaging the life and death of mRNAs in single cells
Dr Jeffrey Chao, Friedrich Miescher Institute for Biomedical Research, Switzerland
After transcription, an mRNA's fate is determined by an orchestrated series of events (processing, export, localisation, translation and degradation) that is regulated both temporally and spatially within the cell. In order to more completely understand these processes and how they are coupled, it is necessary to be able to observe these events as they occur on single molecules of mRNA in real-time in living cells. To expand the scope of questions that can be addressed by RNA imaging, we are developing multi-color RNA biosensors that allow that status of a single mRNA molecules (e.g. translation or degradation) to be directly visualised and quantified.
In order to image the first round of translation, Dr Chao has developed TRICK (translating RNA imaging by coat protein knock-off) which relies on the detection of two fluorescent signals that are placed within the coding sequence and the 3′UTR. In this approach, an untranslated mRNA is dual labeled and the fluorescent label in the coding sequence is displaced by the ribosome during the first round of translation resulting in translated mRNAs being singly labeled. A conceptually similar approach was used for single-molecule imaging of mRNA decay, where dual-colored mRNAs identify intact transcripts, while a single-colored stabilized decay intermediate marked degraded transcripts (TREAT, 3′ RNA end accumulation during turnover). Dr Chao is using these tools to characterise localised translation and degradation during normal cell growth and stress.
Paradoxical role for ribosomes in Ago2-mediated mRNA cleavage
Marvin Tanenbaum, Hubrecht Institute, The Netherlands
Guided by a small RNA, Argonaute 2 (Ago2) interacts with its target mRNA, resulting in mRNA cleavage and decay. In vitro measurements have provided important insights in the binding and cleavage kinetics of Ago2, but much less is known about the behavior of Ago2 in living cells. Here, Marvin describes a method based on his previously developed SunTag translation imaging system, which allows him to observe cleavage of single mRNAs by Ago2 in living cells. He found that Ago2 frequently cleaves its target mRNA within minutes after nuclear export, at a time that precisely coincides with the arrival of the first translating ribosome at the Ago2 binding site. Using translation drugs and different mRNA reporters, he showed that ribosomes can stimulate mRNA decay by Ago2, by promoting the release of cleaved mRNA fragments from Ago2. However, the role of ribosomes in modulating Ago2 activity is paradoxical, as ribosomes also inhibit mRNA cleavage by Ago2, by displacing Ago2 molecules from the mRNA before cleavage can occur. Whether ribosomes promote or inhibit cleavage depends on mRNA release kinetics of the small RNA/Ago2 complex, and Ago2 showed distinct release kinetics when associated with different small RNAs. In summary, through live-cell single molecule imaging, he found that ribosomes profoundly alter Ago2’s mRNA cleavage activity by modulating distinct rate constants of the Ago2 cleavage cycle.
3'UTR-mediated protein-protein interactions regulate protein functions
Dr Christine Mayr, Memorial Sloan Kettering Cancer Center, USA
At least half of human genes use alternative cleavage and polyadenylation to generate mRNA transcripts that differ in the length of their 3' untranslated regions (3'UTRs) while producing the same protein. We found that 3'UTRs can mediate protein-protein interactions, and thus, can determine protein localization and protein functions. Dr Mayr showed that the long 3'UTR of CD47 is required for the formation of a protein complex between CD47 and SET. This interaction enables CD47 protein to efficiently localise to the cell surface, but it also changes protein function as only CD47 protein that was generated from the long 3'UTR isoform (CD47-LU) is able to activate the Ras GTPase RAC1.
Dr Mayr investigated the mechanism of 3'UTR-dependent interaction between SET and CD47 and found that the RNA-binding protein TIS11B is necessary for this process. TIS11B is known to bind to AU-rich element (ARE) containing mRNAs and destabilizes specific mRNAs. Using super-resolution live cell imaging, we observed that TIS11B assembles into a large tubule-like meshwork that is intertwined with the endoplasmic reticulum (ER). The TIS11B granules enrich membrane protein-encoding mRNAs that contain multiple AREs in their 3'UTRs, including CD47-LU, and they exclude mRNAs lacking these features. The TIS11B granules also enrich specific proteins including chaperones, but they exclude SET. The spatial arrangement of the TIS11B granules and the ER enables the ER to act as diffusion boundary for SET. This traps SET at the ER surface and facilitates 3'UTR-mediated protein-protein interactions between SET and newly made membrane proteins, including CD47-LU and PD-L1.
The function of TIS11B in the destabilization of mRNAs does not require TIS11B granule formation. Therefore, when RNA-binding proteins are soluble or present as single entities, they can regulate mRNA-based processes, including mRNA stability. However, assembly of RNA-binding proteins into larger aggregates, including RNA granules, allows them to acquire new properties that are necessary for the regulation of protein functions through 3'UTR-dependent protein-protein interactions.