Characterization of ncRNAs' post-transcriptional modifications by antisense affinity capture and MS analysis

项目摘要 该提案旨在建立一个综合分类平台， 非蛋白质编码RNA（ncRNA）中转录后修饰（PTM）的表征。的 用于检测N6-甲基腺苷（m6 A）的方便方法的可用性，该方法依赖于 特异性抗体和RNA-seq，使突破性的研究，揭示了意义， 这一PTM在必要的监管过程中。特别是关于病毒复制和可卡因的开创性报告 成瘾已经清楚地表明，m6 A途径是非常有前途的目标，为发展新的 治疗策略一种基于质谱的多功能方法的实现 让我们发现许多RNA病毒的基因组是由不同类型的RNA修饰的 除了M6 A之外，还有其他的修改。我们还发现长链非编码RNA参与应激反应， 和癌症都含有核糖核苷酸变体的星座。根据m6 a的先例，我们预计 阐明它们在各自亲本RNA活动中的作用将打开无数新的 治疗干预的途径。 该项目将开发用于确定经后乳腺癌发病率和分布的工具， 这对于阐明其功能至关重要。反义DNA探针文库将用于捕获 通过遗传筛选鉴定的所需RNA类别，将立即分析PTM 内容遵循分而治之的策略，图书馆将逐步缩小池的目标， 能够对携带PTM的RNA进行分类。通过本发明提供的多重样品富集， 捕获过程将允许单个RNA的分离和浓缩，以进行质量分析。 作图和测序。样品制备步骤将在微量滴定孔板上进行， 多路复用整个过程，减少样品损失，支持无人值守操作，最大限度地减少 每个分析的持续时间，并消除连续分析之间的任何延迟。 平台开发最初将采用由合成和重组组成的标准， RNA将被来自不同细胞系统的实际生物样本迅速取代。的 后者将主要由从人单核细胞和酵母培养物获得的长的非编码RNA组成 在不同的压力条件下。这些结果将为PTM在以下方面的作用提供新的宝贵见解： 人类和酵母中由同源p38-MAPK和HOG途径介导的应激反应， 分别这里开发的使能技术将立即使几个正在进行的 在遗传学、表观遗传学、人类病毒学和癌症生物学领域的合作。的能力 把这些项目带到新的难以想象的方向，证实了这些项目的卓越创新影响。 技术.这些项目代表了更广泛的社区， 了解PTM对其亲本RNA结构和功能的影响。基于这个理由， 这些技术将成为任何健康领域的重要研究和诊断工具 涉及RNA调节的病症，包括不同类型的癌症，神经缺陷， 发育畸形、生长和智力迟钝、线粒体疾病和易感性 病毒感染和压力

Project Summary This proposal aims at the development of a platform for the comprehensive classification and characterization of post-transcriptional modifications (PTMs) in non-protein coding RNAs (ncRNAs). The availability of a convenient approach for the detection of N6-methyladenosine (m6A), which relies on specific antibodies and RNA-seq, has enabled groundbreaking studies that revealed the significance of this PTM in essential regulatory processes. In particular, seminal reports on viral replication and cocaine addiction have clearly shown that m6A pathways are very promising targets for the development of new therapeutic strategies. The implementation of a more versatile approach based on mass spectrometry allowed us to show that the genome of many RNA viruses is decorated by different types of RNA modifications in addition to m6A. We also found that long non-coding RNAs involved in stress response and cancer contain constellations of ribonucleotide variants. Based on the m6A precedents, we anticipate that elucidating their roles in the activities of the respective parent RNAs will open countless new avenues for therapeutic intervention. This project will develop tools for determining the incidence and distribution of PTMs, which is essential for their functional elucidation. Libraries of antisense DNA-probes will be employed to capture desired classes of RNAs identified by genetic screens, which will be immediately analyzed for PTM content. Following a divide-and-conquer strategy, the libraries will target progressively narrower pools to enable the classification of PTM-bearing RNAs. The multi-fold sample enrichment afforded by the capture process will allow the isolation and concentration of individual RNAs to be submitted to mass mapping and sequencing. The sample preparation steps will be carried out on microtiter well plates to multiplex the entire process, reduce sample losses, support unattended operations, minimize the duration of each analysis, and eliminate any delay between consecutive analyses. The platform development will initially employ standards consisting of synthetic and recombinant RNAs, which will be promptly replaced with actual biological samples from different cellular systems. The latter will primarily consist of long non-coding RNAs obtained from human monocytes and yeast cultures under different stress conditions. The results will provide new precious insights into the role of PTMs in the stress response mediated in humans and yeast by the homologous p38-MAPK and HOG pathways, respectively. The enabling technologies developed here will immediately benefit several ongoing collaborations in the fields of genetics, epigenetics, human virology, and cancer biology. The ability to take these projects in new unimaginable directions substantiates the excellent innovative impact of these technologies. These projects are representative of much broader communities with an enormous stake in understanding the effects of PTMs on the structure and function of their parent RNAs. For this reason, these technologies will be poised to become an essential research and diagnostic tool for any health condition involving RNA regulation, including different types of cancers, neurological deficits, developmental malformations, growth and mental retardation, mitochondrial disorders, and susceptibility to viral infection and stress.