Characterization of Alu element expression and A-to-I RNA editing in mammals

Cattenoz, M. Pierre Barnabe (2012). Characterization of Alu element expression and A-to-I RNA editing in mammals PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Cattenoz, M. Pierre Barnabe
Thesis Title Characterization of Alu element expression and A-to-I RNA editing in mammals
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor John Mattick
Eric Westhof
Total pages 165
Total colour pages 39
Total black and white pages 126
Language eng
Subjects 060102 Bioinformatics
060802 Animal Cell and Molecular Biology
Formatted abstract
The Short INterspersed Element (SINE) Alus are the most prolific retrotransposons in human. With over one million copies, they comprise more than 10% of the human genome and these non-autonomous retrotransposons spread by hijacking the transposition machinery of the autonomous retrotransposon Long Interspersed Element (LINE1). As a response to retrotransposon invasion, organisms developed mechanisms to preserve the integrity of their genome, and one of them is RNA editing. RNA editing is the modification of one or more bases of an RNA molecule. The most abundant type of editing in mammals is A-to-I editing where the ADAR family transforms adenosine into inosine. The main targets of the ADARs in human are the Alu elements. Editing of the Alu elements targets them to the paraspeckles, thus lead to their sequestration in the nucleus which prevents their interaction with the transposition machinery of LINE1. Editing of Alu elements also mutates their internal POLIII promoter and their poly-A tail, thus preventing their subsequent transposition.

The current view on Alu elements is that they are mainly dormant occupants of the genome. In the first part of this study, we challenge this view by characterizing their activity. After demonstrating that Alu element transcripts can be precisely identified on a large scale with current deep-sequencing technology, the primary sequences of Alu elements are screened for active internal RNA polymerase III promoter by screening POLIII-CHIPseq data. The length and identity of the Alu transcripts are then determined in the cytoplasm and nucleus of cell as well as their association with polysomes and chromatin by screening deep-sequencing data performed on each one of these cell compartments. Analysis of a transcriptome Atlas of 16 human tissues reveals that Alu elements transcription is a widespread phenomenon in normal tissues which correlates with functional LINE1 elements expression. This suggests that Alu element retrotransposition may be a natural mechanism in most normal human tissues. Further analyses show that SINE and LINE expression in somatic tissues is not exclusive to human but also occurs in mouse. Finally, attempts are made to identify tissue specific insertions in the human genome resulting from retrotransposition events.

In the second part of this study, a new method is developed to understand the full impact of RNA editing on Alu transcripts and more broadly on whole transcriptomes by characterizing the edited RNA in a high-throughput fashion. In a first unsuccessful attempt, immunoprecipitation was used to pull-down RNA associated with the editing enzymes ADARs. Further fruitless attempts were then made to pre-purify the complex RNA-ADAR by nuclear fractionation or sucrose gradient before immunoprecipitation. Finally, instead of using an antibody-based approach targeting the ADAR proteins, a protocol targeting directly the inosine in the RNA molecule was developed. First, the RNA is sequestered on magnetic beads. Then an inosine specific cleavage based on RNAseT1 treatment of RNA protected with glyoxal and borate allows the separation of the edited RNA from the total RNA. Finally, deep sequencing is used to identify edited RNA. 1,822 editing sites are found by this method including 28 new editing sites modifying the coding sequences of genes and editing in rRNA, snoRNA and snRNA which were never observed before.
Keyword RNA
Retrotransposons
Deep-sequencing
ADAR
SINE
Alu Elements
Editing
Inosine

 
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Created: Mon, 19 Nov 2012, 23:45:41 EST by Pierre Cattenoz on behalf of Scholarly Communication and Digitisation Service