Over the last funding period, we have made a paradigm-shifting observation: that the information contained in the promoter dictates the fate of the mRNA in the cytoplasm. Hence mRNA fate in the cytoplasm can be determined at the time of its birth. This remote control regulation of the mRNA outcome appears to apply to genes with time dependent expression patterns, such as cell cycle, metabolic product or stress induced genes. We propose to uniquely integrate biochemical, genetic and imaging approaches to identify the hierarchy of factors imprinted co-transcriptionally in the mRNA as well as to define the regulatory DNA sequences that dictate this mRNA behavior. The use of innovative technology, combining the use of microscopy with fluorescent tagging of mRNA is directed toward elucidating the coordination of transcription and downstream events of the RNA life; events that likely create an autoregulatory loop. We envision a tight coupling from transcription through decay and back, mediated by specific factors that would be released from the mRNA upon its degradation, to signal the gene to make more. We hypothesize that cell cycle and stress-regulated genes require a robust coordination between transcription, translation and decay. We expect that factors imprinted by the promoter regulate the strength of the transcriptional response. Taken together, these observations suggest a shuttling of factors between the nucleus and the cytoplasm important for fine-tuning the response of the cell.
The Aims of this project chart a straightforward path to investigate the mechanisms regulating the fate of an mRNA during transcription and how the cells achieve precise gene expression patterns by coordinating mRNA transcription, translation and decay. In order to do this, we will develop and use tools specifically reporting each of the steps in mRNA metabolism and identify factors that coordinate these steps. 1. Impact of promoter sequence on transcription and mRNA localization 2. Purification of factors regulating transcription and mRNA fate 3. Visualization of proteins loaded on the nuclear transcripts and followed into the cytoplasm 4. Kinetics of translation and decay and factors
PROJECT RELEVANCE Despite years of research, we still do not understand how the cell regulates the appearance and disappearance of messenger RNAs (mRNA) and proteins necessary for the cycles of cell growth and division. We have discovered that correct regulation relies on tagging the mRNAs responsible for cell division proteins with the seeds of its own destruction at the moment of its birth. These tags then destroy the mRNA at the exact moment its job is finished, so that the cell can progress to the next part of the cycle by making new proteins. We have developed new imaging tools that will let us see this happen in living cells in real time in order to understand how the cell orchestrates this synchrony. Cell growth and division are the bases of all biological processes, normal and diseased and this understanding will provide information for addressing runaway cell growth such as in cancer, or flaws in cell division that may lead to birth defects or susceptibility to infections.
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|Campbell, Philip D; Chao, Jeffrey A; Singer, Robert H et al. (2015) Dynamic visualization of transcription and RNA subcellular localization in zebrafish. Development 142:1368-74|
|Grimm, Jonathan B; English, Brian P; Chen, Jiji et al. (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods 12:244-50, 3 p following 250|
|Hocine, Sami; Vera, Maria; Zenklusen, Daniel et al. (2015) Promoter-Autonomous Functioning in a Controlled Environment using Single Molecule FISH. Sci Rep 5:9934|
|Wu, Bin; Miskolci, Veronika; Sato, Hanae et al. (2015) Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences. Genes Dev 29:876-86|
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