Uncovering the Role of RNA Modifications in the Paraspeckle

揭示 RNA 修饰在副斑斑中的作用

• 批准号: 10679290
• 负责人: Lauren Wilson
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679290
• 关键词: A549; Adenosine; Affect; Area; Biology; Biophysics; CRISPR interference; Cancer Prognosis; Cancer cell line; Cell Line; Cells; Cellular biology; Characteristics; Chemicals; Complement; Data; Enzymes; Excision; Fluorescence Recovery After Photobleaching; Fluorescence-Activated Cell Sorting; Fluorescent in Situ Hybridization; Gene Expression; Goals; HIV-1; HL60; Hela Cells; Human; Hydrophobicity; Immunofluorescence Immunologic; Induced Mutation; Literature; Location; MALAT1 gene; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Membrane; Messenger RNA; Methods; Microscopy; Modification; Mutate; Nuclear; Oncogenes; Patient-Focused Outcomes; Pattern; Process; Protein Isoforms; Proteins; Proteomics; RNA; RNA-Directed DNA Polymerase; RNA-Protein Interaction; Reader; Regulation; Reporter; Reporting; Research; Resolution; Ribosomal RNA; Role; Sampling; Site; Stress; Structure; System; TP53 gene; Techniques; Therapeutic; Time; Transcript; Transfer RNA; Tumor Suppressor Proteins; Untranslated RNA; Validation; Work; base; cell type; crosslink; detection method; experimental study; genome wide screen; genome-wide; insight; interest; mutant; novel; novel therapeutic intervention; overexpression; scaffold; stoichiometry; transcriptome; transcriptome sequencing; tumor; tumor progression

PROJECT SUMMARY RNA modifications are chemical changes made to transcripts that can regulate their processing, structure, and stability. Recent advances in modification detection techniques have revealed the presence of RNA modifications in mRNA and lncRNA, expanding the known regulatory potential of these modifications beyond their canonical roles in tRNA and rRNA. RNA modifications have been found to modulate the expression of oncogenes and tumor suppressors alike, demonstrating the need to better understand the basic mechanisms of this process so that specific and effective cancer therapeutics can be developed. A critical gap in the literature is the spatial context of modified transcripts; many studies use RNA from whole cells and may miss key mechanisms by averaging the effects of RNA modifications across the transcriptome. I will bring a new perspective to RNA modification biology by focusing my work on a single subcellular context: the paraspeckle. Paraspeckles are stress-inducible nuclear bodies that are assembled on the lncRNA NEAT1, and both this transcript and the paraspeckle itself have been implicated in human cancers. I have used mass spectrometry and sequencing to identify novel RNA modifications on NEAT1, and I hypothesize that these and other modifications on NEAT1 contribute to the assembly of functional paraspeckles. Critically, my preliminary results indicate that the modification profile of NEAT1 differs between cell lines, so I will perform experiments in lines from two cancers marked by overexpression of NEAT1 and one where NEAT1 is downregulated, so that I can look for common mechanisms as well as patterns in the differences between them. In Aim 1, I will focus on NEAT1 directly. I propose the expansion of my current mass spectrometry and sequencing methods so that I can assemble a more complete map of RNA modifications on NEAT1, including the validation and quantification of modifications at single-base resolution. I will mutate identified modification sites, then measure the effects on NEAT1 stability and isoform distribution by qPCR and effects on protein interactions through crosslinking and proteomic analysis. In Aim 2, I will investigate the paraspeckle. I will use both an unbiased genome-wide screen using a paraspeckle reporter system and a targeted microscopy screen of known RNA modification enzymes to identify novel regulators of the paraspeckle. I will make catalytically inactive mutants of the top hits from these screens and perform modification-sensitive RNA sequencing to determine whether these enzymes are modifying NEAT1, other components of the paraspeckle, or upstream regulators, and use fluorescence recovery after photobleaching to measure modification-specific changes in paraspeckle dynamics. Together, these aims will discover and characterize RNA modifications that have a role in paraspeckle formation, revealing insights in an unexplored area of RNA cell biology.

项目总结 RNA修饰是对转录本进行的化学改变，可以调节其加工、结构和 稳定性。修饰检测技术的最新进展揭示了RNA的存在 在mRNA和lncRNA中的修饰，将这些修饰的已知调控潜力扩大到 它们在tRNA和rRNA中的典型作用。已发现RNA修饰可以调节HSP70的表达 癌基因和肿瘤抑制基因相似，表明有必要更好地了解基本机制 这样就可以开发出特效的癌症治疗方法。一个关键的差距， 文献是修饰转录本的空间背景；许多研究使用的是整个细胞的RNA，可能会遗漏 通过平均整个转录组中RNA修饰的影响来实现关键机制。我会带来一辆新的 通过将我的工作集中在一个单一的亚细胞背景上：副斑点，透视RNA修饰生物学。 Paraspeckles是组装在lncRNA NEAT1上的应力诱导的核体，这两个 转录本和副斑点本身与人类癌症有关。我用了质谱仪 和测序以确定NEAT1上的新的RNA修饰，我假设这些和其他 NEAT1上的修饰有助于功能副蛋白的组装。关键是，我的初步结果 表明不同细胞系的NEAT1的修饰谱不同，所以我将在细胞系中进行实验 从两种以NEAT1过度表达为标志的癌症和一种NEAT1下调的癌症中，这样我就可以 在它们之间的差异中寻找共同的机制和模式。在目标1中，我将重点放在 直接使用NEAT1。我建议扩展我目前的质谱学和测序方法，以便我 可以在NEAT1上组装更完整的RNA修改图，包括验证和 以单碱基分辨率量化修改。我会突变已识别的修饰位点，然后测量 定量聚合酶链式反应对NEAT1稳定性和异构体分布的影响及其对蛋白质相互作用的影响 交联和蛋白质组学分析。在目标2中，我将研究副斑点。我会用两个不偏不倚的 使用准斑点报告系统和已知RNA的靶向显微镜筛选进行全基因组筛选 修饰酶以鉴定副斑病的新调节剂。我会制造出催化失活的突变体 并执行修饰敏感的RNA测序以确定 这些酶正在修饰NEAT1，副斑点的其他成分，或上游调节因子，并使用 光漂白后的荧光恢复以测量副视斑的修饰特异性变化 动力学。总之，这些目标将发现和表征RNA修饰，这些修饰在 副斑点形成，揭示了RNA细胞生物学中一个尚未探索的领域的见解。