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: 7993494
• 负责人: Terunaga Nakagawa
• 金额: $ 29.81万
• 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/7993494
• 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）：我们星球上的生命起源被广泛认为是所谓的“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，世界上充满了具有自我复制酶能力的 RNA。 RNA世界的历史记录在今生。例如，核糖体是一种肽键形成酶，其催化核心仅由RNA形成。核糖体中的蛋白质具有相当辅助和调节作用，这些作用是在进化过程中后来获得的。小RNA是证明RNA在各种生物过程中重要调节功能的另一个例子。脂质膜是如何加入RNA世界的？细胞膜在为细胞质中的化学反应提供理想条件方面发挥着极其重要的作用。然而，没有令人信服的模型可以解释“RNA世界”之后膜是如何融入生命的。在这个 EUREKA 提案中，我将检验以下假设：存在某种形式的 RNA 可以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RNA调节质膜的功能。在另一种情况下，可能存在由RNA与辅助蛋白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 在膜上发挥作用的系统的一个例子。总而言之，RNA 很有可能嵌入膜中并在生物学中发挥根本性的重要功能。为了验证这一假设，我们将研究是否有任何 RNA 形式是从脑膜中共纯化的。大脑将被用作模型器官，因为它含有丰富多样的膜。将采取两种方法； (1)我们将生化富集神经元膜并化学剥离外周膜附着蛋白。我们将用去污剂溶解这些膜，并通过将 RNA 与跨膜蛋白分离来分离它们。 (2)来自脑的总RNA将被重组为由总脑脂质构成的膜。通过密度梯度超速离心将膜与未结合的 RNA 分离。分离的膜将溶解在去污剂中并进一步重构为脂质体。通过反复重复脂质重构、分离和溶解，我们将富集膜结合的 RNA。我们将确定已识别 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 或核糖体 RNA。如果我们能够成功识别出这种在膜中发挥作用的新型 RNA 形式，我们将进一步确定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实体，这将彻底改变我们描述生物学和医学的方式。特别是，由于大脑的脂质成分是所有器官中最高的，我们预计这项研究的结果将强烈影响对神经系统生理和功能障碍的理解。 公共健康相关性：该提案旨在识别细胞膜中新型 RNA，这些 RNA 具有根本上重要的生物学功能，例如离子通道、转运蛋白和膜结构调节剂的功能。这种 RNA 的发现可以解释富含脂质的器官（例如大脑）膜中 RNA 介导的新现象。由于脂质代谢和膜形态的功能障碍已经与多种疾病有关，因此该项目获得的结果可能会加深我们对多种疾病的理解，包括脆性X智力低下、精神分裂症、自闭症和痴呆症。