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的探索景观。最近， 在暴露于人类细胞时，施坦兹实验室鉴定了约2,000个长的非编码（LNC）RNA，这样 作为热量，高盐和氧化应激，而其他人则证实了它们对病毒感染，癌症和 老化。当RNA聚合酶II失败时，被称为“基因下游”的狗，这些LNCRNA会导致这些LNCRNA 为了清除新生的RNA 3'在注释位点结束，以作为蛋白质编码基因的子集，我们称为“父母” 基因”。相反，转录从5到45 kbps继续下游，狗保留在 核。狗RNA在压力时在数分钟的时间尺度上表达，这表明它们是“首先 响应者”以帮助细胞生存。总狗占所有基因成绩单的15％–30％，但不是 甚至在人类基因组中被注释。综上所述，这些功能定义了迫切需要确定 狗RNA的顺序和功能，这是该提案的核心目标。在AIM 1中，我们建议对 使用新兴的长读测序方法，从5'到3'末端的单个狗都建立了 Neugebauer实验室中的Polya+和Polya -RNA。我们将利用身体压力来通过命令诱导狗 在几个平台上的大小和优化图书馆准备，以实现适当的测序 我们旨在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RNA被剪接，修饰和聚腺苷酸化的程度。重要的是，我们将测试 我们的工作假设基于初步结果，这些结果是剪接和Hisstone翻译后修改 在狗生物发生中发挥机械作用。这些发现将为我们提供有关 应力途径撞击的细胞机械。在AIM 2中，我们提出了同时的功能分析 利用我们最近的初步发现，小鼠干扰素β基因产生狗的狗会增强 暴露于聚元（模仿病毒感染的模仿）时，干扰素β随后表达。因此，我们会的 询问其他狗在暴露于第二个压力后同样是其父母基因的主要表达。我们 将追求其他初步结果，表明狗的父子基因与转录相关 抑制作用和狗的产量有可能向上或下调父母基因。我们会证明 通过转录伸长和染色质景观的分析，狗的作用机理 狗基因区域具有新的和已发表的芯片数据。最后，确定狗的半恋之前，在 在压力之后，我们将使我们能够探索狗是存储库的概念新颖的可能性 未经处理的预MRNA随后成熟，在从压力中恢复过程中成为活跃的mRNA。这 这些目标的实现将阐明全新的NCRNA类的序列和功能，如 以及转录活性受细胞调节的基因区域和染色质环境 压力。此外，可能会鉴定出完全新颖的基因调节途径。