This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Regulation of gene expression takes place at many steps in the process of converting a gene to an active protein capable of performing its cellular function. Regulatory events that affect the stability and translation of individual mRNA transcripts are crucial for important biological processes ranging from embryogenesis (maternal RNA storage) to neuronal function (Fragile X mental retardation). However, work from many laboratories has only begun to uncover the relevant mechanistic details and environmental or developmental signals. Many important post-transcriptional regulatory events take place in dynamic cytoplasmic domains known as P-bodies. P-bodies are sites of RNA storage and degradation that can also modulate mRNA translation: RNA transcripts are translationally silenced while stored in the P-body and can be subsequently degraded or released back into the translating pool. However, the signals for determining mRNA localization to P-bodies, degradation, or release have not been identified. Using fluorescence microscopy in live yeast cells, we found that a sequence-specific RNA-binding protein, Puf3 (as a Puf3-GFP fusion), co-localizes with P-bodies (as tdimer2 RFP fusion proteins) under specific environmental conditions. This finding suggests that RNA binding proteins, like Puf3, may serve regulatory signals targeting specific sets of RNA transcripts to one or more of the processes associated with P-bodies. We hope to extend these results using fluorescence microscopy to further characterize the environmental conditions and the protein factors required for the co-localization of Puf3 and a handful of other RNA binding proteins. We would also like to examine the effects of environmental conditions and protein factors on the localization of Puf3-dependent mRNAs to P-bodies.
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