Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Papavasiliou Laboratory


Post-transcriptional modifications such as alternative splicing have been shown to add to the complexity needed to compensate for the relatively low number of genes found in higher organisms. Many other modifications recently found in mRNA, which cannot be deduced from what is coded in the genome, such a cytidine to uridine and adenosine to inosine editing, reveal that this complexity is ever expanding. Therefore, the current challenge is to understand what is the function of these modifications. In this thesis, I focus on APOBEC1-mediated RNA editing. In higher eukaryotes RNA editing consists of C to U and A to I transitions mediated by the proteins of the APOBEC1 and ADAR families, respectively. APOBEC1 has been fully characterized in its role of editing the Apolipoprotein B (ApoB) transcript in the intestine, where the C to U modification changes a glutamate codon to a stop codon, creating a smaller version of the ApoB protein. Editing of ApoB is essential for the formation of the chylomicron, a lipid transport protein, making APOBEC1 a crucial enzyme for lipid metabolism. In recent years, our laboratory developed a comparative approach that uses an Apobec1-deficient mouse in order to find true Apobec1-mediated editing events in wildtype mRNA. This method allowed for the discovery of additional sites in a transcriptome-wide manner. Using this, we were able to identify hundreds of additional edited sites in murine intestine and macrophages. This opens up the possibility of alternative APOBEC1 functions. In this thesis, I focus on the edited sites within macrophages, where Apolipoprotein B is not expressed and therefore, where APOBEC1 may play an alternative role to lipid metabolism. Specifically, these consist of 410 highconfidence C to U editing events contained in 275 transcripts, the large majority of which are within the 3'UTR. First, finding that there are no transcriptional differences between macrophages derived from Apobec1-deficient and wildtype mice, I characterized the fate of these transcripts at the molecular level. Previously, ADAR editing has been shown to potentially regulate transcript abundance by nuclear retention and stabilization. Here I demonstrated that this is not the case for APOBEC1. However, even though at the RNA level, there seem to be no alterations due to editing, I showed that editing does regulate translation of protein products, some of which are miRNA mediated. The changes observed at the protein level are nevertheless quite small, which is to be expected from the low frequency of editing per transcript. However, a very longstanding question in the field is whether this is the reflection of a few cells within the population that possess 100% editing per site or whether each cell has low frequency editing. In order to test for this, we created a statistical model that tested the variability of editing at each site among many cells of the same population. Using this model, we observed that cells are indeed quite variable in terms of editing. Then I validated the results of the model using barcodes that identify individual RNA molecules to amplify a region surrounding the edited sites in single cells. Altogether, these experiments demonstrated that within the population, cells that are seemingly transcriptionally identical are indeed heterogeneous. Next, I tested whether the loss of this variability might affect the activity of macrophages. To do this, I designed in vitro and in vivo assays. I demonstrated that Apobec1-deficient macrophages have altered migration and phagocytosis phenotypes in vitro. This predicts that the physiology of monocytes in the Apobec1-deficient mouse would be altered. While setting up a competitive reconstitution in vivo assay, where I would be able to test wildtype and Apobec1-deficient monocytes side by side, I discovered that the development of monocytes is not equivalent between the two genotypes. Surprisingly, Apobec1-deficient monocytic progenitors tend to outcompete their wildtype counterparts and monocytes have an increased preference to form a proinflammatory Ly6C positive phenotype. Finally, I present a possible link between loss of Apobec1 and brain disorders.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Included in

Life Sciences Commons