nai

Project Progress

One of the fundamental properties of cellular life is individuation, or compartmentalization. For example, a genome must exist in its own compartment (for example, inside a membrane) in order for the genome to benefit from its own gene expression. Without some such compartmentalization, which associates a genotype with its phenotype, there would be no possibility of Darwinian evolution. In an RNA world, the possible interactions of lipid bilayers (the likely compartment boundary) and RNA (likely the only encoded macromolecule early on) would therefore be crucial to evolutionary progress. Accordingly, we have been studying RNAs that interact with phospholipid bilayers, and these studies have been supported under this project in the last Annum.

1. We have found that cellular RNA sequences that are localized to membrane systems in a living cell (Xlsirts and VegT mRNA localization signals from the tree frog, Xenopus) will bind to liposomes in vitro, when added as pure transcripts to vesicles made of chemically pure lipids. Furthermore these binding reactions occur at physiological ionic strength (binding is ionic, and declines as ionic strength increases). The binding half-times are of the order of minutes. Thus RNAs transcribed in vitro can reside for significant times on membranes under cellular solution conditions, in an experimental context (membranes made of chemically pure lipids) in which proteins can play no role in membrane affinity. These experiments show for the first time that modern cellular RNAs can have an intrinsic affinity for membrane surfaces. They do not comprise a convincing argument that these RNAs function this way in cells, but show that this is possible because the hypothetical interaction between RNAs and membranes is measurable in test tube experiments.
2. We have taken a step toward the cellular experiment by showing that the same RNAs can be injected into oocytes, whereupon they bind to membrane systems. This experiment was dome with RNAs made fluorescent by an intercalated dye, and the fluor could be seen directly on the cellular membranes. Even more convincing, a dye in the membrane itself showed FRET (Fluorescent Resonance Energy Transfer) to the dye in the RNA, showing that they were very closely associated as expected for a direct interaction.
3. We have completed the construction of a transcribable genomic library of human DNA, in order to make an unbiased search in the genome for RNAs that have membrane affinity.