Biogenesis and function of a novel class of stress-induced long non-coding RNAs

一类新型应激诱导的长非编码RNA的生物发生和功能

• 批准号: 10158039
• 负责人: Karla M Neugebauer
• 金额: $ 41.19万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-19 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158039
• 关键词: Achievement; Address; Aging; Architecture; Back; Biogenesis; Canis familiaris; Cell Nucleus; Cell physiology; Cells; Cellular Stress; Chromatin; Chromosomes; Cleaved cell; Code; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Disease; Environment; Exons; Exposure to; Failure; Feedback; Feeds; Foundations; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genetic Variation; Genomic approach; Goals; Health; Heat Stress Disorders; Heat-Shock Response; Histones; Human; Human Genome; Individual; Infection; Interferon-beta; Interferons; Introns; Knowledge; Learning; Length; Libraries; Malignant Neoplasms; Mediating; Messenger RNA; Methodology; Methods; Modeling; Modification; Molecular; Mouse Cell Line; Mus; Mutation; Nuclear; Nuclear RNA; Outcome; Oxidative Stress; Parents; Pathway interactions; Pattern; Physiological; Play; Poly A; Post-Translational Protein Processing; Preparation; Production; Proteins; Publishing; RNA; RNA Processing; RNA Splice Sites; RNA Splicing; RNA analysis; RNA chemical synthesis; Recording of previous events; Recovery; Regulation; Research; Role; Site; Sodium Chloride; Stress; Structure; Technology; Testing; Therapeutic; Transcript; Transcription Elongation; Transcription Initiation Site; Untranslated RNA; Viral Cancer; Virus Diseases; Withdrawal; Work; base; cell type; experience; exposed human population; first responder; functional genomics; gene repression; genomic tools; insight; knock-down; mRNA Precursor; novel; physiologic stressor; repository; response; therapeutic RNA; transcriptome; transcriptome sequencing

ABSTRACT/PROJECT SUMMARY The non‐coding (nc) transcriptome remains an under‐explored landscape for functional genomics. Recently, ~2,000 long non‐coding (lnc)RNAs were identified by the Steitz lab upon exposure of human cells to stress, such as heat, high salt and oxidative stress, while others have confirmed their induction in viral infection, cancer and aging. Called DoGs for “Downstream of Gene” transcripts, these lncRNAs result when RNA polymerase II fails to cleave nascent RNA 3' ends at the annotated site for a subset of protein‐coding genes that we term “parent genes”. Instead, transcription continues from 5 to 45 kbps further downstream, and DoGs are retained in the nucleus. DoG RNAs are expressed on the timescale of minutes upon stress, suggesting they are among the “first‐ responders” to help cells survive. Total DoGs account for 15%–30% of all intergenic transcripts, yet they are not even annotated in the human genome. Taken together, these features define an urgent need to determine the sequence and function of DoG RNAs, which are central goals of this proposal. In Aim 1, we propose to sequence individual DoGs from their 5' to 3' ends, using emerging long read sequencing methodology established for polyA+ and polyA‐ RNA in the Neugebauer lab. We will exploit physiological stresses to induce DoGs by orders of magnitude and optimize library preparation on several platforms to achieve the appropriate sequencing length and depth for all of the parameters we aim to quantify. The data will reveal the actual lengths, 5' and 3' ends and the extent to which DoG RNAs are spliced, modified and polyadenylated. Importantly, we will test our working hypotheses based on preliminary results that splicing and histone post‐translational modifications play mechanistic roles in DoG biogenesis. These findings will give us the first concrete clues regarding the cellular machineries impinged upon by stress pathways. In Aim 2, we propose concurrent functional analyses of DoGs that exploit our recent preliminary finding that DoG production by the mouse interferon‐β gene enhances subsequent expression of interferon‐β upon exposure to polyIC (mimic of viral infection). Therefore, we will ask whether other DoGs likewise prime expression of their parent genes upon exposure to a second stress. We will pursue other preliminary results suggesting that DoG parent genes are associated with transcriptional repression and that DoG production has the potential to up‐ or down‐regulate the parent gene. We will probe the mechanism of action of DoGs through analyses of transcription elongation and the chromatin landscape in DoG gene regions with new and published ChIP data. Finally, determination of DoG half‐lives before, during and after stress will allow us to explore the conceptually novel possibility that DoGs are repositories for unprocessed pre‐mRNAs that are later matured to become active mRNAs during recovery from stress. The achievement of these aims will illuminate the sequences and function(s) of an entirely new class of ncRNA, as well as the gene regions and chromatin environments where transcriptional activity is regulated by cellular stresses. Moreover, entirely novel lncRNA‐mediated pathways of gene regulation are likely to be identified.

摘要/项目摘要 非编码(NC)转录组仍然是功能基因组学中一个未被充分探索的领域。最近， Steitz实验室在人类细胞暴露于应激时识别出约2,000个长的非编码(LNC)RNA，如 如高温、高盐和氧化应激，而其他已证实它们会导致病毒感染、癌症和 衰老。被称为“基因下游”转录物的狗，当RNA聚合酶II失效时，这些lncRNAs就会产生 为了切割新生的RNA3‘末端的蛋白质编码基因子集的注释位置，我们称之为“亲本 基因“。取而代之的是，转录继续从5到45kbps进一步下游，狗被保留在 原子核。狗的RNA是在压力下几分钟的时间尺度上表达的，这表明它们是最早的 响应者“来帮助细胞存活。狗占所有基因间转录物的15%-30%，但它们不是 即使在人类基因组中也有注解。综上所述，这些功能定义了确定 狗RNA的序列和功能，这是这项提案的中心目标。在目标1中，我们建议对 个体狗从5‘端到3’端，使用为以下目的建立的新兴长阅读测序方法 Neugebauer实验室的Polya+和Polya-RNA。我们将利用生理应激来诱导狗的命令 并在多个平台上优化文库准备，以实现适当的测序 我们要量化的所有参数的长度和深度。数据将揭示实际长度，5‘和3’ 末端和狗的RNA被剪接、修饰和多腺化的程度。重要的是，我们将测试 我们的工作假设基于初步结果，剪接和组蛋白翻译后修饰 在狗的生物发生中发挥机械作用。这些发现将为我们提供关于 受到应力路径冲击的细胞机械。在目标2中，我们提出了并发泛函分析 利用我们最近的初步发现，通过小鼠干扰素-β基因产生的狗增强了 随后干扰素-β在接触PolyIC(模拟病毒感染)时的表达。因此，我们将 问问其他狗是否同样在第二次应激时启动了它们的亲代基因的表达。我们 将继续寻求其他的初步结果，表明狗的父母基因与转录 抑制和狗的生产有可能上调或下调亲本基因。我们会调查 从转录延伸和染色质景观分析狗的作用机制 狗基因区域与新的和公布的芯片数据。最后，确定狗的半衰期之前、期间 在压力之后，我们可以探索狗作为储藏库的概念上的新可能性 未处理的前信使核糖核酸，在从压力中恢复的过程中，随后成熟成为活性信使核糖核酸。这个 这些目标的实现将阐明一类全新的ncRNA的序列和功能(S)，如 以及转录活性受细胞调控的基因区域和染色质环境 压力。此外，可能会发现全新的、由lncRNA介导的基因调控途径。