Molecular Mechanisms of Co-Transcriptional Ribonucleoprotein Assembly

共转录核糖核蛋白组装的分子机制

• 批准号: 10331029
• 负责人: Margaret Louise Rodgers
• 金额: $ 8.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2022-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10331029
• 关键词: Award; Bacteria; Binding; Binding Proteins; Biochemistry; Biogenesis; Biological Models; Cells; Collaborations; Complement; Complex; Coupled; Coupling; DNA Polymerase I; Disease; Dissection; Eukaryota; Event; Fellowship; Gene Expression; Genetic Diseases; Genetic Transcription; Goals; High-Throughput Nucleotide Sequencing; In Vitro; Individual; Kinetics; Light; Malignant Neoplasms; Measures; Mentors; Methods; Modeling; Modification; Molecular; Molecular Chaperones; Molecular Machines; Monitor; Multiplexed Analysis of Projections by Sequencing; Mutation; Nucleotides; Pathway interactions; Phase; Play; Process; Proteins; RNA; RNA Binding; RNA Folding; RNA Probes; RNA chemical synthesis; RNA-Protein Interaction; Resolution; Ribonucleoproteins; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Small Nucleolar Ribonucleoproteins; Spliceosomes; Structure; System; Testing; Time; Training; U3 small nuclear ribonucleoprotein; Work; Yeasts; dimethyl sulfate; experience; experimental study; in vivo; insight; particle; prevent; protein complex; recruit; single molecule; success; tool; yeast genetics

PROJECT SUMMARY RNAs are integral components of molecular machines that carry out all essential processes in gene expression. To expand the functional landscape of these molecular machines, RNAs synergize with proteins to form large complexes called ribonucleoproteins (RNPs). RNPs are formed initially during transcription, where synthesis of the RNA is coupled to RNA folding and association of proteins. A possible consequence of this coupling is that improper co-transcriptional folding may delay protein association, thereby hindering RNP assembly. Yet, RNPs like the ribosome form within minutes in the cell suggesting that there are mechanisms to prevent misfolding or slow assembly. The ribosome represents an ideal model system for studying co-transcriptional RNP assembly, because it contains a highly structured RNA that must be properly folded and assembled to function. Decades of studies on bacterial ribosome assembly have supported a model for assembly in which ribosomal protein association is strictly hierarchical; however, recent evidence from my work and others suggests that while stable incorporation may be hierarchical, underlying transient protein binding nonetheless influences the RNA folding path. The mechanism for how proteins chaperone the RNA during transcription to accelerate assembly is currently unclear. Furthermore, while a similar ordered assembly mechanism has been proposed for eukaryotic ribosome assembly, it is likely that underlying protein binding dynamics also plays a role in guiding folding of the RNA during transcription. This proposal aims to understand the molecular consequences that arise from coupling between transcription, RNA folding, and ribosome assembly by measuring RNA folding directly during co- transcriptional assembly (Aim 1) and visualizing protein association on nascent eukaryotic RNAs (Aim 2). To examine RNA folding during transcription-coupled ribosome assembly in Aim 1, I will probe the RNA structure in real time in vitro during transcription using dimethyl sulfate (DMS) mutational profiling with sequencing (DMS- MaPseq). This method will provide a complete picture of the folding pathway while the RNA is being synthesized in the presence and absence of proteins, thereby allowing for dissection of the individual contributions to the assembly mechanism. Studying RNA folding directly will be complemented by single-molecule experiments in Aim 2 that directly examine protein/RNP binding kinetics. Specifically, I will examine binding of UtpA and U3 snoRNP in real time to nascent yeast ribosomal RNA. Transitioning to studying transcription-coupled ribosome biogenesis in eukaryotes will provide new insight into how transient binding may be a common theme in RNP assembly. Results from the K99 phase will be expanded upon in the independent phase to examine folding and assembly of larger yeast assembly intermediates, such as the 5’ external transcribed spacer particle. In total, these aims will advance our understanding of the mechanistic underpinnings of how transcription-coupled RNP assembly occurs normally and shed light on how RNP assembly can be altered in disease.

项目概要 RNA 是执行基因表达所有基本过程的分子机器的组成部分。 为了扩展这些分子机器的功能，RNA 与蛋白质协同形成大分子机器。 称为核糖核蛋白（RNP）的复合物。 RNPs最初是在转录过程中形成的，其中合成 RNA 与 RNA 折叠和蛋白质关联偶联。这种耦合的一个可能的结果是 不正确的共转录折叠可能会延迟蛋白质结合，从而阻碍 RNP 组装。然而，RNP 就像细胞中几分钟内形成的核糖体表明存在防止错误折叠或 组装速度慢。核糖体代表了研究共转录 RNP 组装的理想模型系统， 因为它含有高度结构化的 RNA，必须正确折叠和组装才能发挥作用。几十年 关于细菌核糖体组装的研究支持了一种组装模型，其中核糖体蛋白 关联是严格等级化的；然而，我的工作和其他人的最新证据表明，虽然稳定 掺入可能是分层的，潜在的瞬时蛋白质结合仍然会影响 RNA 折叠 小路。蛋白质在转录过程中陪伴 RNA 加速组装的机制是 目前还不清楚。此外，虽然已经为真核生物提出了类似的有序组装机制 核糖体组装过程中，潜在的蛋白质结合动力学也可能在指导核糖体折叠中发挥作用 转录过程中的RNA。该提案旨在了解耦合产生的分子后果 通过在共聚合过程中直接测量 RNA 折叠，在转录、RNA 折叠和核糖体组装之间进行分析 转录组装（目标 1）和可视化新生真核 RNA 上的蛋白质关联（目标 2）。到 在目标 1 中检查转录偶联核糖体组装过程中的 RNA 折叠，我将探测以下中的 RNA 结构 使用硫酸二甲酯 (DMS) 进行实时体外转录过程中的突变分析和测序 (DMS- 映射序列）。该方法将提供合成 RNA 时折叠途径的完整图像 在存在和不存在蛋白质的情况下，从而可以剖析个体对蛋白质的贡献 装配机制。直接研究 RNA 折叠将得到单分子实验的补充 目标 2 直接检查蛋白质/RNP 结合动力学。具体来说，我将检查 UtpA 和 U3 的结合 snoRNP 实时检测新生酵母核糖体 RNA。转向研究转录偶联核糖体 真核生物的生物发生将为瞬时结合如何成为 RNP 的常见主题提供新的见解 集会。 K99 阶段的结果将在独立阶段进行扩展，以检查折叠和 较大酵母组装中间体的组装，例如 5' 外部转录间隔颗粒。总共， 这些目标将促进我们对转录耦合 RNP 的机制基础的理解 组装正常发生，并揭示了 RNP 组装如何在疾病中改变。