Molecular Mechanisms of Co-Transcriptional Ribonucleoprotein Assembly

项目总结 RNA是分子机器的组成部分，它执行基因表达中的所有必要过程。 为了扩展这些分子机器的功能格局，RNA与蛋白质协同形成大的 称为核糖核蛋白(RNPs)的复合体。RNP最初是在转录过程中形成的，在那里合成 RNA与蛋白质的RNA折叠和结合有关。这种耦合的一个可能的后果是 不正确的共转录折叠可能会延迟蛋白质的结合，从而阻碍RNP的组装。然而，RNPs 例如核糖体在细胞内几分钟内形成，这表明存在防止错误折叠或 组装速度慢。核糖体是研究共转录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 对细菌核糖体组装的研究支持了一种组装模型，在该模型中，核糖体蛋白 关联是严格分级的；然而，来自我的工作和其他人的最近证据表明，尽管稳定 掺入可能是分层的，潜在的瞬时蛋白质结合仍然影响RNA折叠 路径。蛋白质如何在转录过程中陪伴RNA以加速组装的机制是 目前尚不清楚。此外，虽然对于真核生物也提出了类似的有序组装机制 核糖体组装，很可能是潜在的蛋白质结合动力学也在指导折叠过程中发挥作用 转录过程中的RNA。这一提议旨在理解偶联所产生的分子后果。 在转录、RNA折叠和核糖体组装之间的关系。 转录组装(目标1)和显示新生真核RNA上的蛋白质关联(目标2)。至 在Aim 1中检查转录偶联核糖体组装过程中的RNA折叠，我将在 在体外转录过程中使用硫酸二甲酯(DMS)突变图谱和测序(DMS- MaPseq)。这种方法将在合成RNA时提供折叠途径的完整图像 在蛋白质的存在和不存在的情况下，从而允许剖析个体对 装配机构。直接研究RNA折叠将得到单分子实验的补充 目的2，直接检测蛋白质/RNP结合动力学。具体来说，我将研究UTPA和U3的绑定 SnoRNP实时到新生酵母核糖体RNA。向转录偶联核糖体研究过渡 真核生物的生物发生将为瞬时结合如何成为RNP的一个共同主题提供新的见解 集合。K99阶段的结果将在独立阶段进行扩展，以检查折叠和 较大的酵母组装中间体的组装，例如5‘外部转录间隔区颗粒。总的来说， 这些目标将促进我们对转录偶联RNP如何产生的机制基础的理解 组装正常发生，并揭示了RNP组装如何在疾病中发生改变。

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.