Isolation of ribonucleic acids that are attached to the neuronal membrane

描述（申请人提供）：我们这个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，有一个充满rna的世界，这些rna具有自我复制的酶的能力。RNA世界的历史记录在当下的生活中。例如，核糖体是一种肽键形成酶，其催化核心完全由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起着极其重要的作用。然而，目前还没有令人信服的模型来解释膜是如何在“RNA世界”之后融入生命的。在这个EUREKA的提案中，我将测试存在某种形式的RNA调节脂质双分子层功能的假设。更具体地说，我认为存在以下几种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和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在膜上起作用的系统的一个例子。综上所述，rna很有可能嵌入在细胞膜中，并在生物学中发挥着重要的作用。为了验证这一假设，我们将研究是否有RNA形式是从脑膜中共同纯化的。大脑将被用作模型器官，因为它含有丰富多样的膜。将采取两种办法；(1)我们将生化富集神经元膜，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rna与跨膜蛋白分离来分离rna。(2)脑总RNA重组成脑总脂质膜。膜将通过密度梯度超离心从未结合的RNA中分离出来。分离的膜将在洗涤剂中溶解，并进一步重组为脂质体。通过反复重复脂质重构、分离和溶解，我们将丰富膜结合RNA。我们将确定鉴定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或核糖体rna。如果我们能够成功地鉴定出这种在膜中起作用的新型RNA形式，我们将进一步努力确定它们在膜中的精确功能。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，因为大脑是所有器官中脂质成分最高的，我们期望这项研究的结果将对神经系统的生理和功能障碍的理解产生强烈的影响。

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.