Project Outcomes Report

Cellular ribonucleic acid (RNA) is a large population of diverse molecules each playing indispensable role in the life of the cell by participating in practically all stages of gene expression. The metaphor "I see" means "I understand” explains multiple efforts to visualize dynamics of different RNAs within the cells. This task, however, is a challenge due to the transient (unstable) character of these molecules, their low concentrations and difficulties in delivering label inside the cell. It is especially tough to label native unmodified RNAs in live cells. The major goal of this project was to develop such a method for labeling native RNA molecules inside live bacterial cells. We sought to program the cell itself to synthesize all components of a sensor, an RNA-protein (RNP) complex that would become fluorescent in the presence of target RNA. In the framework of this project we successfully developed a new RNA labeling approach that uses two tools: split aptamer approach and protein complementation. We have shown that the new method allows sequence-specifically label target RNA without labeling DNA; we also showed that the signal from RNA is visible using both cell population measurements (FACS) and single cell analysis (fluorescent microscope). In the course of this study we revealed the mechanism of the intracellular fluorescent RNP complex formation that allowed us to rationally design the probes for targeting RNAs. We successfully labeled and visualized a specific bacterial RNA that is involved into the stress response when bacteria are exposed to the low phosphate media. The fluorescent signal was proportional to RNA concentration and has been localized to specific sub-cellular locations that do not overlap with the bulk of DNA. We showed that the method has single molecule resolution.

Thus, the first universal method for labeling native RNAs in live bacterial cells has been developed. This method can be applied to a variety of different RNAs since the process of new probe preparation is simple. Additionally, , the same principle can be used to study RNA in any type of cells using different aptamers and corresponding proteins. Thus, cell biology, biotechnology and system biology received a new tool that is needed to advance RNA studies in natural environment.

Application of the new labeling method to bacterial mRNAs allowed getting new results on their localization. The data on PstC mRNA suggest that this RNA is localized to the sites other than the bulk of nucleoid DNA. These data imply that RNA moved from the transcription site to other sub-cellular location. Thus, these new data extends the list of such localized RNAs and calls for studies on mechanisms of localization. With this method in hands different question on RNAs processing in bacteria can be addressed.

The broader impact of these findings is vast.  The new principle of the RNA-dependent assembly of the RNP complexes can be expanded to other proteins They can be used as indicators of RNA presence and used in diagnostics; they can also be used as a tool for the directed RNA destruction (if RNA is a carrier or linked to a disease). In this case this method can be applied as a therapeutic tool. 

The success of this project is determined by the efficient and productive collaboration between several experts in various fields. The PI assembled a team that included Dr. Smolina (BME, BU), an expert in molecular biology, cloning and in vitro fluorescence measurements, Dr. Ding (Clemson University, SC), an expert in protein and RNA modeling and Dr. Sun (BME, BU) and Dr. Wanunu (Northeastern University), the experts in single molecule measurements.

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