Regulation of mRNA Stability in Plastids - a Genetic and Biochemical Approach

高等植物的grissem质体通过调节基因的转录速率及其mrna的加工和稳定性来响应发育程序和环境信号。质体mRNA稳定性的改变是一个重要的调控机制，对翻译效率有重要的功能影响。大多数质体mRNA在其3‘非翻译区（UTR）有一个倒置重复（IR）序列，这是正确的mRNA 3’端加工和成熟mRNA稳定性所必需的。突变或3' IR序列的缺失会导致mRNA的错误加工和/或快速衰减。已经鉴定出五种核编码蛋白以结构和/或序列依赖的方式与几种质体mrna的3' UTR相互作用。该项目将采用生物化学和遗传学相结合的方法来研究每种蛋白质的功能及其可能的相互作用。使用重组或部分分离的体外系统，将单独或组合测试Al蛋白在mRNA 3'端加工和稳定性中的作用。这些研究将辅以平行的反义rna策略，利用拟南芥来确认蛋白质的体内功能。先前的实验已经表明28RNP对叶片叶绿体发育至关重要。类似的反义rna策略将用于确定24RNP的发育作用，24RNP的表达模式与28RNP不同。100RNP将测试其与24RNP和28RNP形成mRNA加工复合物的潜在相互作用。其他实验将研究29RNP和55RNP的功能以及这些蛋白与petD mRNA的3'UTR的相互作用。rna结合蛋白的生化和遗传解剖将为细胞器基因表达的核控制提供新的信息。由于光合作用对植物生产力的重要性，在这一应用中提出的基础研究可能与未来的植物和农业技术相关。叶绿体的发育和光合能力对植物的生长和功能至关重要。研究表明，质体通过调节质体基因组编码基因的表达来响应植物的发育程序和环境信号。发育过程中质体基因表达的一个重要控制发生在mRNA的加工和稳定性水平上。mRNA稳定性的变化可以直接影响在光合作用和其他叶绿体过程中具有重要功能的蛋白质的翻译。叶绿体mrna的加工和稳定性是由核编码蛋白介导的，这些蛋白与RNA分子中的特定序列或结构相互作用。在先前的研究中，已经确定了五种蛋白质参与叶绿体mrna的加工和稳定性。目前的项目将采用生物化学和遗传学相结合的方法来研究每种蛋白质的功能及其可能的相互作用。总之，叶绿体mRNA稳定机制的生化和遗传学解剖将为细胞器基因表达的核控制提供新的信息。由于光合作用对植物生产力的重要性，本项目提出的基础研究可能与未来的植物和农业技术相关。＊＊＊

9404841 Gruissem Plastids in higher plants respond to the developmental program and environmental signals by adjusting the transcription rate of genes and the processing and stability of their mRNAs. The change in plastid mRNA stability is an important regulatory mechanism that is of functional consequence for translational efficiency. Most plastid mRNAs have an inverted repeat (IR) sequence in their 3' untranslated region (UTR) that is required for correct mRNA 3' end processing and the stability of the mature mRNA. Mutagenesis or deletion of the 3' IR sequence causes incorrect processing and/or rapid decay of the mRNA. Five nuclear-encoded proteins have been identified that interact with the 3' UTR of several plastid mRNAs in a structural and/or sequence-dependent manner. This project will take a combined biochemical and genetic approach to investigate the function of each protein and their possible interactions. Al proteins will be tested individually or in combinations for their role in mRNA 3' end processing and stability using reconstituted or partially fractionated in vitro systems. These studies will be complemented by parallel antisense-RNA strategies using Arabidopsis to confirm the in vivo function of the proteins. Previous experiments have already shown that the 28RNP is critical for chloroplast development in leaves. Similar antisene-RNA strategies will be used to determine the developmental role of the 24RNP which differs in its expression pattern from the 28RNP. The 100RNP will be tested for its potential interaction with the 24RNP and 28RNP to form a mRNA processing complex. Other experiments will investigate the function of the 29RNP and 55RNP and the interaction of these proteins with the 3'UTR of the petD mRNA. Together the biochemical and genetic dissection of the RNA-binding proteins will provide novel information on the nuclear control of organelle gene expression. Because of the importance of photosynthesis for plant productivity, the basic research proposed in this application is likely to have relevance for future plant and agricultural technologies. %%% Development of chloroplasts and photosynthetic competence are critical for plant growth and function. It has been shown that plastids respond to the plant developmental program and environmental signals by adjusting the expression of genes encoded in the plastid genome. One important control of plastid gene expression during development occurs at the level of mRNA processing and stability. Changes in mRNA stability can directly affect the translation of proteins that have essential functions in photosynthesis and other chloroplast processes. Processing and stability of chloroplast mRNAs are mediated by nuclear-encoded proteins that interact with specific sequences or structures in the RNA molecule. In previous studies, five proteins have been identified that participate in the processing and stability of a specific class of chloroplast mRNAs. The current project will take a combined biochemical and genetic approach to investigate the function of each protein and their possible interactions. Together, the biochemical and genetic dissection of the mechanisms by which chloroplast mRNA stability is accomplished will provide novel information on the nuclear control of organelle gene expression. Because of the importance of photosynthesis for plant productivity, the basic research proposed in this project is likely to have relevance for future plant and agricultural technologies. ***