Uncovering the Role of RNA Modifications in the Paraspeckle

项目摘要 RNA修饰是对转录物进行的化学变化，可以调节其加工，结构和功能。 稳定修饰检测技术的最新进展揭示了RNA的存在 mRNA和lncRNA的修饰，扩展了这些修饰的已知调控潜力， 它们在tRNA和rRNA中的典型作用。已经发现RNA修饰可以调节 癌基因和肿瘤抑制基因一样，证明需要更好地了解基本机制 这样就可以开发出特异有效的癌症治疗方法。一个关键的差距， 文献是修饰的转录本的空间背景;许多研究使用来自整个细胞的RNA， 通过平均RNA修饰在转录组中的作用来研究关键机制。我会带一个新的 通过将我的工作集中在一个单一的亚细胞背景上：paraspeckle。 Paraspeckles是在lncRNANEAT 1上组装的应激诱导的核小体，这两个核小体都是由神经元细胞的细胞核组成的。 转录本和旁斑本身与人类癌症有关。我用质谱分析法 和测序，以确定NEAT 1上的新RNA修饰，我假设这些和其他 NEAT 1上的修饰有助于功能性旁斑的组装。关键是，我的初步结果 表明NEAT 1的修饰谱在细胞系之间不同，因此我将在细胞系中进行实验 从两个NEAT 1过表达的癌症和一个NEAT 1下调的癌症中， 在它们之间的差异中寻找共同的机制和模式。在目标1中，我将重点关注 NEAT 1直接我建议扩展我目前的质谱和测序方法， 可以在NEAT 1上组装更完整的RNA修饰图谱，包括验证和 以单碱基分辨率定量修饰。我将突变确定的修改网站，然后测量 通过qPCR对NEAT 1稳定性和亚型分布的影响以及通过 交联和蛋白质组学分析。在目标2中，我将研究paraspeckle。我会用一个公正的 使用paraspeckle报告系统的全基因组筛选和已知RNA的靶向显微镜筛选 修饰酶，以鉴定paraspeckle的新调节剂。我会制造出催化活性不高的突变体 并进行修饰敏感的RNA测序，以确定是否 这些酶修饰NEAT 1、paraspeckle的其他组分或上游调节剂， 光漂白后的荧光恢复以测量旁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， paraspeckle形成，揭示了RNA细胞生物学未探索领域的见解。

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.