Destruction of noncoding RNAs

非编码 RNA 的破坏

• 批准号: 10674858
• 负责人: BENJAMIN MONTEVERDE KLEAVELAND
• 金额: $ 42.38万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10674858
• 关键词: Area; Binding; Binding Sites; Cell Line; Cells; Child; Code; Complex; Dedications; Development; Disease; Engineering; Environment; Gene Expression; Genes; Knowledge; Malignant Neoplasms; Mediating; MicroRNAs; Modeling; Molecular Biology Techniques; Neurons; Organism; Outcome; Pathway interactions; Postdoctoral Fellow; Process; Production; Proteins; RNA; Regulation; Regulator Genes; Research; Resistance; Role; Site; Slice; Stimulus; Synaptic Vesicles; Transgenic Mice; Ubiquitination; Untranslated RNA; Viral; Work; cell type; circular RNA; cis acting element; gene product; genome-wide; improved; infancy; multicatalytic endopeptidase complex; posters; posttranscriptional; recruit; response; ubiquitin-protein ligase; vesicular release

SUMMARY Precise and dynamic control of gene expression is a balancing act between production and destruction. Although noncoding RNAs are critical regulators of gene expression with important roles in development and disease, our understanding of their destruction is still in its infancy. One of the reasons for this gap in knowledge is that some noncoding RNAs, like microRNAs and circular RNAs, are resistant to the known pathways that destroy protein- coding RNAs. Here I present my plan to identify how these two types of noncoding RNA are destroyed. MicroRNAs (miRNAs) are protected from degradation by their effector protein Argonaute. One way to degrade miRNAs is through target-directed miRNA degradation (TDMD), which occurs when a highly complementary viral or artificial RNA interacts with the miRNA. My postdoctoral work identified two of the first examples of endogenous targets that induce miRNA degradation, which also led to our discovery of an E3 ubiquitin ligase that mediates TDMD and the identification of 48 additional miRNAs (likely an underestimate) subject to TDMD. Our working model now is that the binding of highly complementary targets to the Argonaute–miRNA complex leads to recruitment of the E3 ligase, ubiquitination and proteosomal degradation of Argonaute, and release and degradation of the miRNA. With the discovery of TDMD effector proteins and the broad influence of TDMD on miRNA stability, a major gap is identifying the target RNAs that induce TDMD. A related problem is understanding why some targets are better than others. We will address these gaps by answering two questions: 1) Which endogenous RNAs induce miRNA degradation? 2) What are the pairing rules and cis-acting elements that promote TDMD? To do so, we will use transgenic mice, engineered cell lines, genome-wide approaches, and classic molecular biology techniques. Our second area of research is understanding how circular RNAs (circRNA) are degraded. The poster child of post-transcriptional circRNA regulation is Cdr1as, a conserved circRNA that is highly expressed in neurons and limits spontaneous synaptic vesicle release. Cdr1as contains a single near-perfect binding site that can be sliced by Ago2-loaded miR-671 and >70 seed sites for miR-7. Previously, I showed that Cdr1as can be regulated by the independent and cooperative actions of miR-671 and miR-7. How miR-7 induces destruction of Cdr1as is unclear as circRNAs should be resistant to miRNA-mediated deadenylation and decapping. Here, we will answer two questions: 1) How is Cdr1as degraded? 2) How are other unstable circRNAs degraded? The outcomes of our research will be the identification and improved understanding of dedicated pathways for degrading noncoding RNAs. These pathways have potential for broad impact as they are likely employed in diverse cell types and organisms and in response to a variety of stimuli.

总结 基因表达的精确和动态控制是生产和破坏之间的平衡行为。虽然 非编码RNA是基因表达的关键调节因子，在发育和疾病中发挥重要作用，我们的研究表明， 对它们的破坏的理解仍处于起步阶段。造成这种知识差距的原因之一是， 非编码RNA，如microRNA和环状RNA，对已知的破坏蛋白质的途径有抵抗力， 编码RNA。在这里，我提出了我的计划，以确定这两种类型的非编码RNA是如何被破坏的。 microRNA（miRNAs）被其效应蛋白Argonaute保护免于降解。一种降低 miRNA是通过靶向miRNA降解（target-directed miRNA degradation，TDMD），当一个高度互补的 病毒或人工RNA与miRNA相互作用。我的博士后工作确定了两个最早的例子， 诱导miRNA降解的内源性靶点，这也导致我们发现了E3泛素连接酶 介导TDMD和鉴定48个额外的miRNA（可能低估）受TDMD影响。 我们现在的工作模型是，高度互补的靶标与Argonaute-miRNA复合物的结合 导致E3连接酶的募集、Argonaute的泛素化和蛋白体降解，以及Argonaute的释放和 降解miRNA。随着TDMD效应蛋白的发现和TDMD对细胞的广泛影响， miRNA的稳定性，一个主要的差距是识别诱导TDMD的靶RNA。一个相关的问题是理解 为什么有些目标比另一些更好我们将通过回答两个问题来解决这些差距：1） 内源性RNA诱导miRNA降解？2)什么是配对规则和顺式作用元件， 推广TDMD？为此，我们将使用转基因小鼠，工程细胞系，全基因组方法， 经典的分子生物学技术我们的第二个研究领域是了解环状RNA （circRNA）被降解。转录后circRNA调控的典型产物是Cdr 1as，一个保守的 在神经元中高度表达并限制自发性突触囊泡释放的circRNA。cdr 1as包含一个 可被Ago 2负载的miR-671切割的单个近乎完美的结合位点和>70个miR-7的种子位点。 以前，我表明Cdr 1as可以通过miR-671和miR-672的独立和协同作用来调节。 miR-7。miR-7如何诱导Cdr 1as的破坏尚不清楚，因为circRNA应该对miRNA介导的 去腺苷化和脱帽。在这里，我们将回答两个问题：1）Cdr 1as是如何降解的？2)好吗 其他不稳定的circRNA降解了吗我们的研究成果将是识别和改进 了解降解非编码RNA的专用途径。这些途径具有广泛的潜力， 影响，因为它们可能用于不同的细胞类型和生物体，并响应于各种刺激。