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

基本信息

批准号：
9981761
负责人：
Thomas Rivas
金额：
$ 3.45万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
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/9981761
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 metabolism RNA-Directed RNA Polymerase Regulation Retrotransposition Role Route Scheme Short Interspersed Nucleotide Elements Site Stress System Transcription Repressor 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

项目摘要

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.

项目摘要 RNA聚合酶II（POL II）使用使用DNA依赖性RNA聚合酶（DDRP），使用使用双链DNA合成蛋白质编码的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作为合成RNA的模板。一个 POL II RDRP活动的示例是非编码B2 RNA的3'末端扩展 B2（EB2）RNA。 B2 RNA由短散布的元素（罪）编码，这些元素存在于350,000多个由于逆转录置置而导致的小鼠基因组中的副本。将这些元素随机插入基因组可能是有害的，具体取决于残疾的区域或基因，在某些情况下可能导致疾病。在细胞应力下，从B2罪的非编码B2 RNA的转录大大增加，从而大大增加增加了B2罪的逆转逆转的可能性。 POL II的RDRP活动可以控制水平转录后通过产生EB2 RNA来促进其降解。拟议的工作将识别细胞中3'修改的B2 RNA的所有形式，测量其稳定性，并确定3'结束的扩展 B2 RNA改变了其细胞内定位和逆转录置。 B2 RNA与POL II结合，全球反映转录，并经历POL II依赖性18 核苷酸RDRP扩展形成EB2 RNA。此外，EB2 RNA的半衰期大大降低了与B2 RNA相比。 B2 RNA的EB2 RNA形成被认为是一种自动调节机制克服转录表示并促进EB2 RNA的分离和降解。迄今为止， B2 RNA的大多数表征是通过生化实验的，而不是包含可能改变POL II RDRP活性效率的细胞系统的复杂性。这建议旨在了解RNA聚合酶在转录后如何控制RNA代谢。鉴定由于扩展，加工而导致的细胞3'修饰的B2 RNA物种，并确定应力如何影响这些物种，将开发出B2 RNA特异性测序方案（B2 RNA-SEQ）。量化 B2 RNA物种的木桩，B2 RNA-Seq将与溴化脉冲脉冲耦合。此外，观察B2和EB2 RNA在压力前后的细胞中的定位，RNA荧光原位将使用杂交（鱼）。鱼将与免疫荧光实验耦合以确定 RNA与潜在处理机械之间的共定位。此外，要确定3'扩展细胞应力会影响B2 RNA逆转录的能力，逆转录的细胞培养模型将被使用。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扩展，RDRP调节的降解，细胞定位和细胞应激在生命周期上这个ncRNA。