ADAR1 AND RNAI

Understanding the mechanism behind ADAR1 (Adenosine DeAminase acting on RNA 1) modulation of the RNA interference pathway

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 Obiettivo del progetto (Objective)

'The most common form of editing in mammalian RNA is deamination of adenosine to inosine by ADAR1 and ADAR2. ADAR1p150, an interferon inducible adenosine deaminase, is also known to antagonize RNA interference (RNAi). To date, the mechanism by which this is carried out is unclear. For example, it is unknown if the embryonic lethality of ADAR1 knockout mice is related to antagonism of RNAi. Thus, I propose to study how ADAR1 antagonizes the RNAi pathway and if it interacts directly with RNAi components. Mutants of ADAR1 have been generated in the host laboratory. I propose to examine the activity of each mutant against RNAi in HEK 293 cells. These mutants will allow us to determine if RNA binding, localization or editing by ADAR1 is required for ADAR1 antagonism of RNAi. The effects on each branch of the RNAi pathway, siRNA and miRNA, will be elucidated using advanced techniques including miRNA detecting microArrays, cloning, dual-luciferase, and miRNA/siRNA reporters. Further, classical approaches of protein-protein interaction, and a recently optimized Tap-tagging approach, will be used to reveal if ADAR1 interacts directly with components of the RNAi machinery. This proposal supports collaboration between Dr. Bret Heale and Dr. Mary O’Connell. Dr. Heale received his PhD in June 2006 (USA) for his studies in the RNAi field, specifically related to siRNA and miRNA recognition of target sites, and in the course of his PhD published two first author publications (one in siRNA targeting and one in miRNA targeting) and authored a computer program to predict siRNA efficacy. Dr. Mary O’Connell (UK) has spent nearly twenty years working in the field of RNA editing, specifically examining ADARs. Thus, this proposal embodies the goals of the FP7 “People” work program to bring talented scientists to Europe, further European science, and to strengthen international ties.'

Introduzione (Teaser)

RNA editing has the potential to alter the function of proteins coded for specific action. This could have important meaning for how receptors in the brain and central nervous system ultimately function.

Descrizione progetto (Article)

In mammals, the most common form of ribonucleic acid (RNA) editing, alteration of the sequence of nucleotides, is the conversion of adenosine to inosine. Common in the nervous system, this takes place by deamination where enzymes act as protein catalysts, removing amine groups from molecules. ADAR1 and ADAR2 are just such deaminases.

In RNA interference, small RNA molecules (microRNAs or miRNAs, and small interfering RNAs or siRNAs) can bind to other specific RNAs (mRNA) and either increase or decrease their activity. RNA interference (RNAi) has an important role in defending cells as well as directing gene development and expression in general.

The 'Understanding the mechanism behind ADAR1 (Adenosine DeAminase acting on RNA 1) modulation of the RNA interference pathway' (ADAR1 and RNAi) project aimed to determine if there is an overlapping of RNA editing and RNAi, and if the processes actually antagonise each other.

Researchers performed deep sequencing of small RNAs to determine if after an increase in the editing enzyme's volume and activity there also would be an increase in the editing of miRNAs. The ADAR1 and RNAi study revealed that increased ADAR1 expression actually did not have an effect on miRNAs. The result suggests there is no overall change in miRNA activity following increased RNA editing

However, researchers hypothesised that effects of an increase in ADAR1 expression could be more difficult to detect. To investigate, they proceeded with modification and mutation experiments to discover what was required for an interaction between miRNAs precursors and ADARs to take place. Results showed that the two pathways could antagonise each other but only on certain miRNAs. Also, the project found that this interaction could be independent of enzymatic activity.