POST-TRANSCRIPTIONAL REGULATORY COMPLEX DYNAMICS IN YEAST

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在将基因转化为能够执行其细胞功能的活性蛋白质的过程中，基因表达的调节在许多步骤中发生。影响单个mRNA转录本的稳定性和翻译的调控事件对从胚胎发生(母体RNA储存)到神经元功能(脆性X智力低下)等重要生物学过程至关重要。然而，许多实验室的工作才刚刚开始揭示相关的机械细节和环境或发育信号。许多重要的转录后调控事件发生在被称为P小体的动态细胞质结构域中。P-小体是RNA的储存和降解部位，也可以调节mRNA的翻译：RNA转录本在P-小体中存储时在翻译上被沉默，随后可以被降解或释放回翻译池。然而，决定mRNA定位于P小体、降解或释放的信号尚未确定。在酵母活细胞中使用荧光显微镜，我们发现一个序列特异性的RNA结合蛋白Puf3(作为Puf3-GFP融合)在特定的环境条件下与P-体(作为tdimer2 RFP融合蛋白)共定位。这一发现表明，RNA结合蛋白，如Puf3，可能向与P小体相关的一个或多个过程提供针对特定RNA转录本的调控信号。我们希望用荧光显微镜扩展这些结果，以进一步表征Puf3和其他几个RNA结合蛋白共定位所需的环境条件和蛋白质因素。我们还想研究环境条件和蛋白质因素对Puf3依赖的mRNAs定位到P小体的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for 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.