Isolation of ribonucleic acids that are attached to the neuronal membrane

分离附着在神经元膜上的核糖核酸

• 批准号: 8103245
• 负责人: Terunaga Nakagawa
• 金额: $ 30.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8103245
• 关键词: Autistic Disorder; Binding; Biogenesis; Biological; Biological Process; Biology; Brain; Catalytic Domain; Categories; Cell membrane; Cellular Membrane; Code; Cytoplasm; DNA; Databases; Dementia; Density Gradient Centrifugation; Detergents; Disease; Endoplasmic Reticulum; Enzymes; Evolution; Fragile X Syndrome; Functional disorder; Genome; Genomics; Integral Membrane Protein; Ion Channel; Life; Lipid Bilayers; Lipids; Liposomes; Mediating; Medicine; Membrane; Membrane Lipids; Membrane Proteins; Mental Retardation; Modeling; Morphology; Nervous system structure; Neurons; Nucleic Acids; Organ; Organ Model; Peptides; Peripheral; Physiology; Planets; Play; Proteins; RNA; RNA Binding; Recording of previous events; Research; Ribosomal RNA; Ribosomes; Role; Schizophrenia; Small RNA; System; Testing; chemical reaction; interest; lipid metabolism; macromolecule; novel; public health relevance; reconstitution; research study

DESCRIPTION (provided by applicant): The origin of life on our planet is widely believed to be the so-called "RNA world". During evolution, before DNA and proteins were part of life there was a world full of RNAs that possess self-replicating enzymatic ability. The history of RNA world is recorded in the current life. For example, ribosome is a peptide-bond forming enzyme whose catalytic core is formed exclusively by RNA. The proteins in the ribosomes have rather accessory and regulatory roles that are acquired later during evolution. The small RNA is another example that demonstrates the important regulatory function of RNA in various biological processes. How did lipid membrane join the RNA world? Cellular membranes have extremely important roles in providing the ideal conditions for the chemical reactions in the cytoplasm. However there is no convincing model that explains how membranes were integrated into life after the "RNA world". In this EUREKA proposal, I will test the hypothesis that some form of RNA exists that regulates the function of lipid bilayers. More specifically, I consider the existence of the following kind of RNAs. First, there may be a category of small RNA that regulates the function of plasma membrane. In another case, there may be primitive ion channels that are formed by RNA with accessory proteins. Protein conducting channels in the endoplasmic reticulum binds to ribosomes and therefore may be considered as one example of a system in which RNAs function at the membrane. Taken together there is a good chance that RNAs are embedded in the membrane and play fundamentally important function in biology. To test this hypothesis, we will investigate whether any RNA forms are co-purified from the brain membranes. The brain will be used as a model organ because it contains a rich variety of membranes. Two approaches will be taken; (1) We will biochemically enrich neuronal membranes and chemically strip off peripheral membrane attached proteins. We will detergent solubilize these membranes and isolate RNAs by separating them from transmembrane proteins. (2) The total RNA from brain will be reconstituted into membrane made of total brain lipids. The membranes will be separated from the unbound RNA by density gradient ultracentrifugation. The isolated membrane will be solubilized in detergent and further reconstituted into liposomes. By iteratively repeating lipid reconstitution, isolation, and solubilization, we will enrich membrane bound RNA. We will determine the sequence of the identified RNAs and search for the genomic database to verify that they are not protein coding RNAs nor ribosomal RNAs. If we will be successful in identifying such novel RNA forms that function in the membrane we will further pursue to define their precise functions in the membrane. The identification of RNAs in the membrane will add yet another entity of biological macromolecules that will revolutionize the way we describe biology and medicine. In particular, because brain has the highest lipid composition of all organs, we expect that the results of this research will strongly impact the understanding of the physiology and dysfunction of the nervous system. PUBLIC HEALTH RELEVANCE: This proposal aims to identify novel form of RNAs in the cellular membrane that possess fundamentally important biological functions such as those of ion channels, transporters, and structural regulators of membrane. A discovery of this kind of RNAs may explain novel phenomena mediated by RNA in the membranes in organs that are rich in lipids, such as brain. Because dysfunction of lipid metabolism and membrane morphology have been already implicated in various disorders, the results obtained form this project may deepen our understanding of a variety of diseases including, fragile-X mental retardation, schizophrenia, autism, and dementia.

描述（由申请人提供）：我们星球上生命的起源被普遍认为是所谓的“ RNA世界”。在进化过程中，在DNA和蛋白质成为生命的一部分之前，有一个充满RNA具有自我复制酶促能力的世界。 RNA世界的历史记录在当前的生活中。例如，核糖体是一种肽键形成的酶，其催化核由RNA仅形成。核糖体中的蛋白质具有相当相当的配件和调节作用，这些作用是在后期进化过程中获得的。小RNA是另一个例子，证明了RNA在各种生物过程中的重要调节功能。脂质膜如何加入RNA世界？细胞膜在为细胞质化学反应提供理想条件方面具有极为重要的作用。但是，没有令人信服的模型可以解释“ RNA世界”之后的膜如何融入生活中。在本尤里卡（Eureka）的提案中，我将检验以下假设：某种形式的RNA存在调节脂质双层的功能。更具体地说，我认为存在以下类型的RNA。首先，可能有一类小的RNA调节质膜的功能。在另一种情况下，可能存在带有辅助蛋白的RNA形成的原始离子通道。内质网中的蛋白质传导通道与核糖体结合，因此可以被视为RNA在膜上功能的系统的一个例子。综上所述，RNA很有可能被嵌入膜中，并且在生物学中起着重要的重要功能。为了检验这一假设，我们将研究是否从脑膜共纯化任何RNA形式。大脑将被用作模型器官，因为它包含各种膜。将采取两种方法； （1）我们将在生物化学上富集神经元膜，并从化学上剥离外周膜附着的蛋白质。我们将通过将这些膜与跨膜蛋白分开来清除这些膜和分离RNA。 （2）大脑的总RNA将被重构为由总脑脂质制成的膜。膜将通过密度梯度超速离心与未结合的RNA分离。分离的膜将在洗涤剂中溶解并进一步重构为脂质体。通过迭代重复脂质重构，分离和溶解化，我们将富集膜结合的RNA。我们将确定已识别的RNA的序列并搜索基因组数据库，以验证它们不是蛋白质编码RNA和核糖体RNA。如果我们将成功地识别在膜上起作用的新型RNA形式，我们将进一步追求定义它们在膜中的精确功能。膜中RNA的识别将增加另一个生物大分子的实体，这些实体将彻底改变我们描述生物学和医学的方式。特别是，由于大脑对所有器官的脂质组成最高，因此我们希望这项研究的结果将强烈影响对神经系统的生理和功能障碍的理解。 公共卫生相关性：该提案旨在确定具有根本重要的生物学功能的细胞膜中新型RNA的形式，例如膜的离子通道，转运蛋白和结构调节剂的生物学功能。这种RNA的发现可能解释了由RNA介导的新型现象，这些现象是在富含脂质（例如脑）的器官中的膜中介导的。由于脂质代谢和膜形态的功能障碍已经与各种疾病有关，因此获得的结果可能会加深我们对各种疾病的理解，包括脆弱的X智力低下，精神分裂症，自闭症和痴呆症。