Mass Spectrometry of Ribosomal RNA:Protein Interactions

描述（由申请人提供）：我们对转录后修饰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（rnas）的核苷，很大程度上受到缺乏能够识别和表征其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发展适当的质谱方法，以表征RNA和RNA-蛋白质相互作用方面的核糖体结构。这项更新的目标是开发新的和改进的质谱（MS）方法来鉴定和表征核糖体内rna -蛋白质相互作用，并应用这些方法来获得有关核糖体结构和功能的生物学相关信息。第一个目标将集中于交联鉴定和测序。这些进展将用于鉴定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。第二个目标将集中在识别和量化伪尿嘧啶。这些进展将用于定量表征23S rRNA中存在的假尿嘧啶。这些拟议研究的意义包括生物分析质谱的发展，用于鉴定和测序rna -蛋白（或dna -蛋白）交联，以及具有位点特异性的假尿嘧啶的定量测定。该研究计划将获得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伪尿苷合成酶rud的作用模式。提出的研究计划中的创新包括开发用于核酸-蛋白质交联选择性鉴定的液相色谱-电感耦合等离子体质谱（LC-ICP-MS），使用电子转移解离（ETD）对寡核苷酸的肽段进行测序：肽异偶联，这导致了一种强大的MS/MS交联测序方法，并创建了假尿嘧啶的定量测定方法。这项研究计划将产生新的和改进的方法来表征rna -蛋白质相互作用。此外，虽然本研究的技术和方法发展将用于表征核糖体，但许多发展将广泛适用于对表征其他核糖核蛋白复合物，dna -蛋白质复合物或其他核酸系统感兴趣的研究人员。重要的是，这项研究计划将为蛋白质合成提供新的重要工具和信息，这对人类健康至关重要。

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.