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: 10546449
• 负责人: Karla M Neugebauer
• 金额: $ 42.05万
• 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/10546449
• 关键词: Achievement; Address; Aging; Architecture; Back; Biogenesis; Cell Nucleus; Cell Survival; Cell physiology; Cells; Cellular Stress; Chromatin; Chromosomes; Code; Data; Disease; Environment; Exons; Exposure to; Failure; Feedback; Feeds; Foundations; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Order; Genes; Genetic Transcription; Genetic Variation; Genomic approach; Goals; Health; Heat Stress Disorders; Heat-Shock Response; Histones; Human; Human Cell Line; 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; Osmosis; Outcome; Oxidative Stress; Parents; Pathway interactions; Pattern; Physiological; Play; Poly A; Polyadenylation; Polymerase; Post-Translational Protein Processing; Preparation; Production; Proteins; Publishing; RNA; RNA Polymerase II; RNA Processing; 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; 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/项目总结 非编码（nc）转录组仍然是功能基因组学的一个未开发的领域。最近， Steitz实验室在将人类细胞暴露于应激后鉴定了约2，000个长非编码（lnc）RNA，例如 如热，高盐和氧化应激，而其他人已经证实其在病毒感染，癌症和 衰老这些lncRNA被称为“基因下游”转录物，当RNA聚合酶II失败时， 在我们称之为“亲本”的蛋白质编码基因亚组的注释位点切割新生RNA 3'末端 基因”。相反，转录在下游从5 kbps继续到45 kbps，并且DoG保留在下游 原子核DoG RNA在应激时以分钟为时间尺度表达，这表明它们是“第一批”表达。 “帮助细胞存活。总的DoG占所有基因间转录物的15%-30%，但它们不是 甚至在人类基因组中也有注释。总之，这些特征表明迫切需要确定 DoG RNA的序列和功能，这是本提案的中心目标。在目标1中，我们建议 从其5'至3'末端，使用建立用于测定单个DoG的新出现的长读段测序方法， polyA+和polyA-RNA在纽介堡实验室。我们将利用生理压力诱导狗的订单 并在几个平台上优化文库制备，以实现适当的测序 长度和深度的所有参数，我们的目标是量化。数据将揭示实际长度，5'和3' 末端和DoG 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，剪接和组蛋白翻译后修饰 在DoG生物发生中起机械作用。这些发现将为我们提供有关 细胞机制受到压力途径的影响。在目标2中，我们提出了并发功能分析， 利用我们最近的初步发现，即小鼠干扰素-β基因产生的DoG增强了 在暴露于polyIC（病毒感染的模拟物）后随后表达干扰素-β。所以我们会 询问是否其他的狗在暴露于第二压力时同样引发它们的亲本基因的表达。我们 将继续研究其他初步结果，这些结果表明DoG亲本基因与转录相关， 抑制，并且DoG的产生具有上调或下调亲本基因的潜力。我们会调查 通过分析转录延伸和染色质景观， DoG基因区与新的和已发表的ChIP数据。最后，确定DoG半衰期之前，期间 和压力后将使我们能够探索概念上新颖的可能性，狗是储存库， 未加工的前mRNA，在从应激中恢复的过程中，其随后成熟成为活性mRNA。的 这些目标的实现将阐明一类全新的ncRNA的序列和功能， 以及基因区域和染色质环境，其中转录活性由细胞调节， 压力此外，可能会发现全新的lncRNA介导的基因调控途径。