Student Theses and Dissertations

Date of Award

2010

Document Type

Thesis

RU Laboratory

Papavasiliou Laboratory

Keywords

RNA editing, APOBEC1 editing targets, ultra-high throughput sequencing, small intestinal enterocytes

Abstract

RNA editing is generally defined as the alteration of an RNA sequence from that encoded by the genome through nucleotide insertion, deletion or modification. The Apolipoprotein B mRNA Editing Catalytic polypeptide 1 (APOBEC1) cytidine deaminase is an mRNA editing enzyme that modifies a specific cytidine in the apolipoprotein B (apoB) transcripts of small intestine enterocytes. APOBEC1-mediated cytidine to uridine editing generates an inframe stop codon and results in translation of a truncated apoB isoform with distinct functions in lipid transport. Other physiological mRNA targets of APOBEC1 editing have remained largely unknown. This thesis presents the development of an RNA-Seq method for the identification of mRNA editing events on a transcriptome-wide scale and its application to the discovery of APOBEC1 editing targets in small intestinal enterocytes. The technique utilizes ultra-high throughput sequencing and subsequent bioinformatic analysis to compare wild-type and congenic apobec1-/- transcriptomes for specific single nucleotide variants indicative of editing. Following technical validation, the screening approach was used to identify more than 30 previously undescribed APOBEC1 editing sites in enterocyte mRNA. All of the newly identified sites are located in AU-rich segments of transcript 3’ untranslated regions (3’ UTRs). Furthermore, these sites share several characteristic sequence features, including flanking nucleotide preferences and a downstream (3’) APOBEC1 mooring motif. Sequence pattern recognition analysis based on these features successfully predicted additional APOBEC1 targets. The studies detailed here demonstrate the feasibility and utility of a novel transcriptomics approach to RNA editing studies. The corresponding results indicate that APOBEC1 site-specifically edits many mRNA transcripts other than apoB in small intestinal enterocytes, suggesting additional roles for APOBEC1 beyond its function in apolipoprotein regulation.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/399

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