Phase Separation as a Regulation Mechanism for Cephalopod ADAR Localization and Function

Abstract

One of the most prevalent causes of protein diversification in complex organisms today is RNA editing, or modifying RNA post-transcriptionally to be translated into different proteins. One method for modifying RNA is deamination, or the process of removing an amino group from an RNA base to change its base pairing properties, thus changing the codon it is recognized as. ADAR enzymes, or adenosine deaminase (RNA specific) enzymes, are enzymes responsible for hydrolytically deaminating site-specific adenosine bases into inosine. These enzymes are found in all mitochondrial eukaryotes but are especially active in cephalopods such as squids and octopi, which boast the most complex nervous systems of all invertebrates. Temperature is a major regulator of this editing activity, as cold-dwelling cephalopods exhibit higher ADAR activity than warm-dwelling cephalopods. ADAR enzymes have been found to interact with the RNA-binding protein hnRNPA1, which forms liquid-liquid phase separated droplets comprising RNA and RNA-binding proteins. Lower temperatures have also been found to promote phase-separated protein cluster formation, which suggests that phase separation could be a regulation factor of ADAR enzymatic activity as ADAR could localize into protein clusters with RNA to increase deamination activity. Therefore, in this thesis the effect of phase separation on ADAR enzymatic activity was measured. First, localization of ADAR isoforms was thoroughly characterized both in the nucleus and cytoplasm of imaging cells. Then, the clustering ability of ADAR at different temperatures was measured, where it was found that ADAR is capable of self-organizing into protein droplets at lower temperatures. Phase diagrams at six different temperatures were constructed to measure ADAR clustering activity with a light-activated Corelet system, and it was found that as temperature decreases, ADAR more readily phase separates. Finally, RNA-seq light activation enzymatic activity assays were carried out at two different temperatures to measure the effect of protein clustering and temperature on ADAR enzymatic activity on a KV1.1 RNA substrate, where it was found that the presence of phase separation and lower temperature provides the optimal condition for A-to-G site editing. Through this work, we can better understand the role of phase separation in organizing and localizing functional compartments that can regulate enzymatic activity in the cell cytoplasm.