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.

项目摘要 RNA修饰是对转录本的化学变化，可以调节其处理，结构和 稳定。修饰检测技术的最新进展揭示了RNA的存在 MRNA和LNCRNA的修改，扩大了这些修饰的已知调节潜力 他们在tRNA和rRNA中的规范作用。已经发现RNA修饰可调节 癌基因和肿瘤抑制剂都表明需要更好地了解基本机制 在此过程中，可以开发特定有效的癌症治疗剂。一个关键的差距 文献是修改后的成绩单的空间上下文。许多研究使用全细胞的RNA，可能会错过 通过平均RNA修饰在转录组中的效果来实现关键机制。我会带一个新的 通过将我的工作集中在单个亚细胞上下文上：The Paraspeckle。 拼接是组装在lncrna neat1上的应力诱导核体，这既 成绩单和拼接本身与人类癌症有关。我使用了质谱法 并进行测序以识别Neat1上的新RNA修饰，我假设这些和其他 对Neat1的修改有助于功能拼贴的组装。至关重要的是我的初步结果 表明Neat1的修改曲线在细胞系之间有所不同，因此我将在线路中执行实验 来自由Neat1过表达的两个癌症和一个neat1下调的癌症，以便我可以 寻找共同的机制以及它们之间差异的模式。在AIM 1中，我将重点放在 Neat1直接。我提出了当前质谱和测序方法的扩展，以便我 可以在Neat1上组装RNA修改的更完整的映射，包括验证和 单基分辨率的修改定量。我将突变确定的修改位点，然后测量 qPCR对NEAT1稳定性和同工型分布的影响以及通过 交联和蛋白质组学分析。在AIM 2中，我将调查拼贴。我将同时使用公正的 使用拼贴报告系统和已知RNA的靶向显微镜屏幕，全基因组屏幕 修饰酶以鉴定羊角面包的新调节剂。我将使催化不活跃的突变体 这些屏幕的最高命中量并执行对修改敏感的RNA测序，以确定是否是否 这些酶正在修改Neat1，拼贴的其他组件或上游调节器，并使用 光漂白后的荧光恢复以测量拼贴的特异性变化 动力学。这些目标共同发现并表征了在 拼贴会形成，揭示了RNA细胞生物学未开发的区域中的见解。