How cell stress and 3' end alterations control the metabolism of a cellular non-coding RNA

细胞应激和 3 末端改变如何控制细胞非编码 RNA 的代谢

• 批准号: 9763333
• 负责人: Thomas Rivas
• 金额: $ 3.4万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9763333
• 关键词: Affect; Amanitins; Base Sequence; Binding; Biochemical; Biological Assay; Bromouridine; Cell Culture Techniques; Cells; Cellular Stress; Code; Complex; Coupled; DNA; DNA Insertion Elements; DNA Polymerase II; DNA Polymerase III; DNA-Directed RNA Polymerase; Data; Development; Disease; Dissociation; Enzymes; Fluorescent in Situ Hybridization; Frequencies; Genes; Genetic Transcription; Genome; Genomics; Half-Life; Heat-Shock Response; Herpesvirus 1; Homeostasis; Human; Immunofluorescence Immunologic; In Vitro; Individual; Infection; Introns; Lead; Life Cycle Stages; Malignant Neoplasms; Mammals; Measures; Messenger RNA; Metabolism; Modeling; Mus; NIH 3T3 Cells; NIH Mouse; Nucleotides; Physiologic pulse; Population; Property; Proteins; RNA; RNA Binding; RNA-Directed DNA Polymerase; RNA-Directed RNA Polymerase; Regulation; Retrotransposition; Role; Route; Scheme; Short Interspersed Nucleotide Elements; Site; Stress; System; Transcription Repressor/Corepressor; Untranslated RNA; Untranslated Regions; Virus Diseases; Work; base; ds-DNA; experimental study; gene repression; inhibitor/antagonist; insight; mouse genome; novel; prevent; promoter; transcription factor; transcriptome sequencing

PROJECT SUMMARY RNA Polymerase II (Pol II) canonically acts as a DNA-dependent RNA polymerase (DdRP), using double stranded DNA to synthesize protein-encoding mRNAs and some non-coding RNAs. Pol II also has RNA-dependent RNA polymerase activity (RdRP), which uses an RNA as a template to synthesize RNA. An example of Pol II RdRP activity is the 3’ end extension of the non-coding B2 RNA to generate extended B2 (eB2) RNA. B2 RNA is encoded by Short Interspersed Elements (SINEs), which exist in over 350,000 copies in the mouse genome due to retrotransposition. Random insertion of these elements into the genome could be deleterious, depending on which region or gene is disrupted, and in some cases can cause disease. Upon cellular stress, transcription of non-coding B2 RNAs from B2 SINEs is greatly increased, thereby increasing the likelihood of retrotransposition of B2 SINEs. The RdRP activity of Pol II could control the levels of B2 RNA post-transcriptionally by generating eB2 RNA to promote its degradation. The proposed work will identify all forms of 3’ modified B2 RNAs in cells, measure their stabilities, and determine how 3’ end extension of B2 RNA changes its intracellular localization and retrotransposition. B2 RNA binds to Pol II, globally represses transcription, and undergoes a Pol II-dependent 18 nucleotide RdRP extension to form eB2 RNA. Furthermore, eB2 RNA has a drastically reduced half-life as compared to B2 RNA. The formation of eB2 RNA from B2 RNA is thought to be an autoregulatory mechanism to overcome transcriptional repression and promote the dissociation and degradation of eB2 RNA. To date, the majority of the characterization of B2 RNA has been through biochemical experiments, which do not encompass the complexity of a cellular system that could alter the efficacy of Pol II RdRP activity. This proposal aims to understand how an RNA polymerase controls RNA metabolism post-transcriptionally. To identify cellular 3’ modified B2 RNA species due to extension, processing, and determine how stress affects these species, a B2 RNA-specific sequencing scheme (B2 RNA-seq) will be developed. To quantify the stabilities of B2 RNA species, B2 RNA-seq will be coupled to bromouridine pulse-chase. Furthermore, to observe the localization of B2 and eB2 RNAs in cells before and after stress, RNA Fluorescence in situ hybridization (FISH) will be utilized. FISH will be coupled to immunofluorescence experiments to determine colocalization between RNAs and potential processing machinery. Additionally, to determine how 3’ extension and cellular stress affect the ability of B2 RNA to retrotranspose, a cell-culture model of retrotransposition will be used. Altogether, these studies will provide an understanding of the interplay between B2 RNA expression, Pol II RdRP extension, RdRP-regulated degradation, cellular localization, and cellular stress on the life cycle of this ncRNA.

项目概要 RNA 聚合酶 II (Pol II) 通常充当 DNA 依赖性 RNA 聚合酶 (DdRP)，使用 双链 DNA 合成蛋白质编码 mRNA 和一些非编码 RNA Pol II 也有。 RNA依赖性RNA聚合酶活性（RdRP），它使用RNA作为模板来合成RNA。 Pol II RdRP 活性的示例是非编码 B2 RNA 的 3' 端延伸，以生成延伸的 B2 (eB2) RNA 由短散布元件 (SINE) 编码，存在超过 350,000 个。 由于这些元件随机插入基因组而导致小鼠基因组中的拷贝。 可能是有害的，具体取决于哪个区域或基因被破坏，并且在某些情况下可能会导致疾病。 在细胞应激时，来自 B2 SINE 的非编码 B2 RNA 的转录大大增加，从而 增加 B2 SINE 逆转录转座的可能性 Pol II 的 RdRP 活性可以控制水平。 拟议的工作将通过生成 eB2 RNA 来促进其降解。 识别细胞中所有形式的 3' 修饰 B2 RNA，测量其稳定性，并确定 3' 末端延伸的方式 B2 RNA 改变其细胞内定位和逆转录转座。 B2 RNA 与 Pol II 结合，全面抑制转录，并经历 Pol II 依赖性 18 核苷酸 RdRP 延伸形成 eB2 RNA 此外，eB2 RNA 的半衰期显着缩短。 与 B2 RNA 相比，B2 RNA 形成 eB2 RNA 被认为是一种自动调节机制。 克服转录抑制并促进 eB2 RNA 的解离和降解。 B2 RNA 的大部分表征都是通过生化实验进行的，而这些实验并没有 涵盖可能改变 Pol II RdRP 活性功效的细胞系统的复杂性。 该提案旨在了解 RNA 聚合酶如何控制转录后的 RNA 代谢。 鉴定由于延伸、加工而产生的细胞 3' 修饰 B2 RNA 种类，并确定应激如何影响 影响这些物种，将开发 B2 RNA 特异性测序方案 (B2 RNA-seq) 来量化。 B2 RNA 物种的稳定性，B2 RNA-seq 将与溴尿苷脉冲追踪相结合。 观察应激前后B2和eB2 RNA在细胞中的定位，RNA原位荧光 将利用杂交 (FISH) 与免疫荧光实验相结合来确定。 另外，RNA 和潜在加工机制之间的共定位，以确定如何进行 3' 延伸。 和细胞应激影响 B2 RNA 逆转录转座的能力，逆转录转座的细胞培养模型将 总之，这些研究将提供对 B2 RNA 表达之间相互作用的理解， Pol II RdRP 延伸、RdRP 调节的降解、细胞定位和细胞应激对生命周期的影响 这个非编码RNA。