Isolation of ribonucleic acids that are attached to the neuronal membrane

描述（由申请人提供）：我们星球上生命的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，有一个充满RNA的世界，这些RNA具有自我复制的酶能力。RNA世界的历史记录在当下的生活中。例如，核糖体是一种形成肽键的酶，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，这些作用是在进化后期获得的。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具有极其重要的作用。然而，没有令人信服的模型来解释“RNA世界”之后膜是如何整合到生命中的。在尤里卡的这份提案中，我将检验这样一个假设，即存在某种形式的RNA来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，可能存在一类调节质膜功能的小RNA。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，因此可以被认为是RNA在膜上发挥功能的系统的一个例子。总的来说，RNA很有可能嵌入细胞膜中，并在生物学中发挥重要的功能。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，因为它含有丰富的各种膜。将采取两种方法：（1）我们将生物化学富集神经元膜和化学剥离外周膜附着蛋白。我们将洗涤剂溶解这些膜，并通过将它们与跨膜蛋白分离来分离RNA。(2)来自脑的总RNA将重构到由总脑脂质制成的膜中。通过密度梯度超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RNA。我们将确定已鉴定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，也不是核糖体RNA。如果我们能够成功地鉴定出这种在膜中发挥作用的新型RNA形式，我们将进一步努力确定它们在膜中的精确功能。细胞膜中RNA的鉴定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，由于大脑在所有器官中具有最高的脂质成分，我们预计这项研究的结果将强烈影响对神经系统生理学和功能障碍的理解。 公共卫生相关性：该提案旨在鉴定细胞膜中具有基本重要生物学功能的RNA的新形式，例如离子通道，转运蛋白和膜结构调节剂。这类RNA的发现可能解释在富含脂质的器官（如脑）中由RNA介导的膜中的新现象。由于脂质代谢和膜形态的功能障碍已经与各种疾病有关，因此从该项目获得的结果可以加深我们对各种疾病的理解，包括脆性X智力低下、精神分裂症、自闭症和痴呆症。

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.