Student Theses and Dissertations

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Darnell Robert Laboratory

Abstract

RNA editing refers to the process by which the sequence of RNA is altered through the insertion, deletion or modification of specific nucleotides. Editing of mRNA transcripts can increase the informational complexity encoded by the genome by producing alternative protein isoforms through specific posttranscriptional RNA editing events. Additionally, RNA editing in non-coding regions of mRNA transcripts has been shown to influence gene expression in a tissue-specific manner. In mammals, mRNA editing serves a diverse set of biological roles in neuronal function, host defense and lipid metabolism. The major mRNA editors acting in mammals include the adenosine deaminases acting on RNA (ADARs) and Apolipoprotein B mRNA Editing Catalytic polypeptide-1 (APOBEC1). The ADARs and APOBEC1 were originally characterized as catalysts for previously characterized biologically important RNA-editing events that resulted in specific coding changes; study of additional editing activity was limited by standard sequencing techniques. APOBEC1 in particular was characterized in the small intestine as mediating a specific editing event in the coding region of Apolipoprotein B (Apob). APOBEC1-dependent RNA editing in Apob mediates the tissue-specific differential expression of Apob isoforms, a process important for intestinal lipid metabolism and transport. The development of next-generation sequencing has allowed for transcriptome-wide discovery of RNA editing activity and has resulted in the identification of more than 10,000 RNA editing events, pointing to more biological functions for RNA editing than had been previously appreciated. To search for additional APOBEC1 editing events, our lab developed a comparative RNA-Seq screen for the transcriptome-wide identification of enzyme-specific RNA editing events. Applying this technique to small intestine enterocytes, the site of known APOBEC1 activity, we identified over 30 novel APOBEC1 editing events in transcript 3’UTRs, which represents the first example of physiological APOBEC1 editing outside of the Apob transcript. These newly identified editing events were located in evolutionarily conserved regions of transcript 3’UTRs, suggesting that this editing activity may have functional relevance. The discovery of additional editing activity for APOBEC1, as well as the fact that it is expressed in a number of immune cell types, suggests that APOBEC1, like other members of the AID/APOBEC family, may contribute to cellular immune processes. The focus of the work presented in this thesis is the identification and characterization of physiological APOBEC1 editing activity in bone marrow derived macrophages (BMDMs). Using a comparative RNA-Seq screen, I identified more than 100 novel APOBEC1 editing events in BMDMs. This APOBEC1 activity occurred in two distinct editing patterns and fell within evolutionarily conserved regions of transcript 3’UTRs. Luciferase reporter assays were utilized to assess the consequences of APOBEC1 3’UTR editing on protein expression and identified a number of combinations of editing events that affect translational outcomes. To determine if APOBEC1 editing could modulate protein expression by altering miRNA targeting, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) of the Argonaute (Ago) proteins was performed on wild-type and APOBEC1-deficient cells. HITS-CLIP yielded transcriptome-wide maps of Ago binding and potential miRNA seed regions. While there was considerable overlap between loci targeted by both Ago and APOBEC1, little evidence was found for APOBEC1 disruption or creation of miRNA seed targets. Overall, this work characterizes abundant APOBEC1 activity in BMDMs that can modulate protein expression levels by a miRNA-independent mechanism. These results point to broader functions for APOBEC1 in transcript regulation and host defense.

Comments

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

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