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.

项目摘要 RNA是分子机器的组成部分，它们在基因表达中执行所有基本过程。 为了扩大这些分子机器的功能景观，RNA与蛋白质协同形成大型 称为核糖核蛋白（RNP）的复合物。 RNP最初是在转录过程中形成的，其中合成 RNA与RNA折叠和蛋白质缔合耦合。这种耦合的可能结果是 不正确的共转录折叠可能会延迟蛋白质关联，从而阻碍RNP组件。但是，RNP 就像细胞中几分钟内的核糖体形式一样，表明有一些机制可以防止错误折叠或 缓慢组装。核糖体代表研究共转录RNP组件的理想模型系统， 因为它包含一个高度结构化的RNA，必须正确折叠并组装以功能。几十年 细菌核糖体组装的研究支持了一种用于组装的模型，其中核糖体蛋白 协会严格是分层的；但是，我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，基本的瞬时蛋白结合仍会影响RNA折叠 小路。蛋白质在转录到加速组装过程中RNA伴侣的机制是 目前不清楚。此外，虽然已经提出了类似的有序装配机制，以实现真核。 核糖体组装，潜在的蛋白质结合动力学也可能在引导折叠中起作用 转录过程中的RNA。该建议旨在了解耦合产生的分子后果 在转录，RNA折叠和核糖体组装之间，通过直接测量RNA折叠 转录组装（AIM 1）和新生真核生物RNA上的蛋白质关联（AIM 2）。到 检查在AIM 1中的转录耦合核糖体组装过程中的RNA折叠，我将探测RNA结构 使用测序使用二甲基硫酸二甲基（DMS）突变分析在转录过程中实时实时（DMS-） MAPSEQ）。该方法将在合成RNA时提供折叠途径的完整图片 在存在和不存在蛋白质的情况下，因此可以解剖个人对该蛋白的贡献 组装机制。直接研究RNA折叠将通过单分子实验完成 AIM 2直接检查蛋白质/RNP结合动力学。具体而言，我将检查UTPA和U3的绑定 实时snonp snornp新生的酵母核糖体RNA。过渡到研究转录耦合核糖体 真核生物中的生物发生将提供新的见解，了解瞬态结合如何成为RNP的常见主题 集会。 K99阶段的结果将在独立阶段扩展，以检查折叠和 较大的酵母组装中间体的组装，例如5'外部转录的间隔粒子。总共 这些目标将提高我们对转录耦合RNP的机械基础的理解 组装正常发生，并阐明了如何在疾病中改变RNP组装。