Mass Spectrometry of Ribosomal RNA:Protein Interactions

描述(申请人提供)：我们对转录后修饰的核苷在蛋白质合成中的功能作用的理解，特别是那些定位于核糖体核糖核酸(RRNAs)的核苷，在很大程度上受到缺乏能够识别和表征它们与蛋白质相互作用的方法的限制。我们研究的长期目标一直是，并将继续开发适当的质谱学方法，以根据RNA和RNA-蛋白质相互作用来表征核糖体的结构。这次更新的目标是开发新的和改进的质谱学(MS)方法来识别和表征核糖体内的RNA-蛋白质相互作用，并应用这些方法来获得关于核糖体结构和功能的生物学相关信息。第一个目标将集中在交联链识别和测序上。这些发展将用于识别参与蛋白质合成起始步骤的RNA和核糖体蛋白的特定识别元件。第二个目标将侧重于识别和量化假尿苷。这些进展将被用来定量描述23S rRNA中存在的假尿苷。这些拟议的研究的意义包括生物分析MS在鉴定和测序RNA-蛋白质(或DNA-蛋白质)交联链方面的进展，以及具有位点特异性的假尿苷的定量测定。这项研究计划将产生关于核糖体的重要生物学信息，包括缺乏Shine-Dalgarno基序的信息在翻译前是如何结合的，核糖体蛋白L31在起始步骤中的作用，以及23S rRNA假尿苷合成酶RluD的作用模式。拟议研究计划中的创新包括开发用于选择性鉴定核酸-蛋白质交联物的液相色谱电感耦合等离子体质谱(LC-ICP-MS)，使用电子转移离解(ETD)对寡核苷酸中的多肽部分进行测序，这导致了用于交链测序的可靠的MS/MS方法，以及创建了一种假尿苷的定量分析方法。这项研究计划将产生表征RNA-蛋白质相互作用的新的和改进的方法。此外，虽然这项研究的技术和方法开发将用于表征核糖体，但许多进展将对有兴趣表征其他核糖核蛋白复合体、DNA-蛋白质复合体或其他核酸系统的研究人员具有广泛的适用性。重要的是，这项研究计划将提供有关蛋白质合成的新工具和重要信息，蛋白质合成对人类健康具有根本意义和相关性。 与公共健康相关：蛋白质合成是生命所必需的基本生物过程。蛋白质合成的部位，核糖体，是由RNA和蛋白质组成的大分子。了解RNA和蛋白质是如何相互作用的，以及它们是如何被修饰的，对于区分细菌核糖体和高等生物(如哺乳动物)的核糖体非常重要。这项研究将创造新的工具，使研究人员能够表征核糖体，这些工具将在这里用于获得与蛋白质合成早期步骤相关的重要信息。

DESCRIPTION (provided by applicant): Our understanding of the functional role of posttranscriptionally modified nucleosides in protein synthesis, especially those localized to ribosomal ribonucleic acids (rRNAs), is limited largely by the lack of methods that can identify and characterize their interactions with proteins. The long-term goal of our research has been and continues to be to develop appropriate mass spectrometric approaches to characterize the structure of the ribosome in terms of RNA and RNA-protein interactions. The goals of this renewal are to develop new and improved mass spectrometry (MS) approaches to identify and characterize RNA-protein interactions within the ribosome, and to apply these methods to obtain biologically relevant information about ribosome structure and function. The first aim will focus on cross-link identification and sequencing. Those developments will be used to identify specific recognition elements of the RNAs and ribosomal proteins involved in the initiation step in protein synthesis. The second aim will focus on identifying and quantifying pseudouridine. Those developments will be used to quantitatively characterize pseudouridines present in 23S rRNA. The significance of these proposed studies include bioanalytical MS developments for identifying and sequencing RNA-protein (or DNA-protein) cross-links and the quantitative determination of pseudouridine with site specificity. This research plan will yield important biological information on the ribosome including how messages lacking the Shine-Dalgarno motif are bound before translation, the role of ribosomal protein L31 in the initiation step, and the mode of action for the 23S rRNA pseudouridine synthase RluD. Innovations in the proposed research plan include developing liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) for selective identification of nucleic acid-protein cross-links, the use of electron transfer dissocation (ETD) for sequencing the peptide moiety in an oligonucleotide:peptide heteroconjugate, which leads to a robust MS/MS method for cross-link sequencing, and creating a quantitative assay for pseudouridine. This research plan will yield new and improved approaches for characterizing RNA-protein interactions. Additionally, while the technology and method developments from this research will be used to characterize the ribosome, many of the developments will have broad applicability for investigators interested in characterizing other ribonucleoprotein complexes, DNA-protein complexes, or other nucleic acid systems. Importantly, this research plan will provide new tools and information of importance about protein synthesis, which is fundamentally significant and relevant to human health. PUBLIC HEALTH RELEVANCE: Protein synthesis is a fundamental biological process required for life. The site of protein synthesis, the ribosome, is a large macromolecule composed of RNAs and proteins. Understanding how RNAs and proteins interact, and how they are modified, is important to differentiate bacterial ribosomes from those of higher organisms, such as mammals. This research will create new tools that enable researchers to characterize ribosomes, and these tools will be used here to obtain important information relating to early steps in protein synthesis.