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.
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.