Spatio-temporal regulation of mRNA decay revealed by a novel single-molecule dual-color imaging method

Discoveries that have been made over past decades emphasized the importance of post-transcriptional control as a means of regulating gene expression. RNA turnover is one of the key aspects of post-transcriptional control that contributes directly towards maintenance of normal cellular homeostasis. Degradation of functional messenger RNAs (mRNAs) is a tightly regulated process and its dysregulation results in either excessive or insufficient amounts of mRNAs within cells that eventually lead to a disease-associated condition. Furthermore, multiple quality control mechanisms eliminate aberrant mRNAs thereby preventing their translation into malfunctioning proteins. The realization of the importance of RNA decay pathways has fueled further research towards understanding the underlying molecular mechanisms in RNA turnover and its regulation.
All protein-coding mRNAs, as well as non-coding RNAs, have distinct half-lives and are ultimately degraded. Previously, many of the factors involved in RNA decay pathways have been identified and studied. Two types of enzymes are shared among RNA decay pathways: exonucleases and endonucleases. The former are further divided into 5′-to-3′ and 3′-to-5′ degrading enzymes and their activation is often dependent on prior removal of terminal stability marks from an RNA molecule. The best-studied exonuclease is Xrn1 that degrades an RNA substrate from the 5′-end to 3′-end. On the other hand, endonucleases cleave an RNA strand to expose the resultant fragments to exonucleases, circumventing the requirement of first removing the stability marks.
Most of our current appreciation of the molecular mechanisms related to the mRNA decay is attributable to the methods that involve ensemble measurements. However, these measurements often result in an averaged outcome from whole population of cells, wherein information about variability among individual cells is lost. In addition, the possibility to get information on the spatio-temporal regulation of mRNA decay is limited using ensemble methods. Hence, accurate dissection of the spatial and temporal regulation of mRNA decay requires development of a single-molecule method that preserves information on cell-to-cell variability. Single-molecule RNA imaging methods have already been used to study several aspects of the mRNA life cycle and they have helped to uncover in vivo regulations that were not possible to observe before. However, a powerful imaging method allowing for an observation of mRNA turnover in real-time at the level of single cells/molecules has been missing.
During my PhD, I established a robust single-molecule imaging technique in order to characterize the spatio-temporal dynamics of RNA turnover within its cellular context. I engineered an mRNA reporter that contains viral tandem pseudo-knots placed between PP7 and MS2 stem-loops. These orthogonal stem-loops can be labeled with spectrally distinct fluorescent proteins. In addition, the viral pseudo-knots block Xrn1-mediated degradation resulting in stabilization of the reporter’s 3′-degradation intermediate that is otherwise inherently instable. This stabilized 3′-end contains only the MS2 stem-loop region. Thus, intact mRNAs are labeled with both fluorophores, while incompletely degraded mRNA fragments are labeled only with a single fluorophore. I used the amounts and positions of intact mRNAs and stabilized 3′-ends as readout of mRNA degradation. Therefore, this technique is called 3(Three)′-RNA End Accumulation during Turnover (TREAT).
I applied TREAT to monitor the fates of mRNAs in single fixed and living mammalian cells. Using this method, I measured the kinetics and cell-to-cell variability of mRNA decay in fixed cells. The nuclear export rates and cytoplasmic mRNA half-lives showed that individual degradation events occur independently within the cytoplasm suggesting that there is no burst in mRNA degradation. In addition, I found that transcripts, as well as degradation intermediates, are dispersed throughout cytoplasm and are not enriched within processing bodies in living cells. Imaging of an mRNA biosensor targeted for an endonucleolytic cleavage by the RNA-induced silencing complex (RISC) showed that slicing can be observed in real-time in cytoplasm of living cells but does not occur in nucleus. The slicing events were found to have no spatial preference with respect to the distance from the nucleus.
In addition to the rate of synthesis and the rate of turnover, the levels of mRNAs were found to be affected by the rate of translation as well. Indeed, I have also observed that inhibition of translation by several compounds increases mRNA stability, suggesting that the processes of mRNA degradation and translation are globally interconnected. The cross-talk among three processes central to the mRNA life cycle, transcription, degradation and translation, is becoming increasingly apparent. However, further research is required to obtain a detailed understanding of the molecular interplays in eukaryotic cells. As TREAT system visualizes mRNA from its synthesis in the nucleus through export to degradation in cytoplasm, I anticipate that this methodology will provide a framework for investigating the entire life history of individual mRNAs in single cells.