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

Final Report Summary - ADAR1 AND RNAI (Understanding the mechanism behind ADAR1 (Adenosine DeAminase acting on RNA 1) modulation of the RNA interference pathway)

The aim of this project was to determine if two RNA pathways: RNA editing and RNA interference, interact and potentially antagonise each other. RNA editing is prevalent in the nervous system and is catalysed by the ADAR proteins. It converts specific adenosines to inosines and has the potential to alter codons and therefore the function of the encoded protein. RNA inference is a method of regulating the abundance of a particular mRNA so that small RNAs (miRNA or siRNA) recognise and base-pair to specific mRNAs. The consequence of this interaction is that either that the mRNA is targeted for degradation or the miRNA prevents the mRNA being translated into protein. Since both RNA editing and RNA interference bind to double-stranded RNA the question was if these pathways would compete for the same substrate and antagonise each other. If this were so then it would have consequences on the functioning of receptors in the brain and central nervous system.

In collaboration with J. Vogel's group in Berlin, we performed deep sequencing of small RNAs from cell lines that had been infected with salmonella and had an induction of ADAR1. We wanted to determine that if there was an increase in the abundance and activity of this editing enzyme would there be a subsequent increase in editing of miRNAs. To our surprise the increase in ADAR1 expression had no affect on miRNAs, they were not edited and their abundance did not change. Therefore this result suggests there is no global alteration in miRNA activity due to increased RNA editing, however the effect of an increase in ADAR1 expression may be more subtle or specific and therefore difficult to detect.

To elucidate if a specific interaction occurred between the two pathways we employed an artificial system. We transfected reporter constructs that respond to alterations in miRNAs level or sequence, into human HEK 293T cells. We choose to study mir-376a2, a known edited miRNA. We also transfected into this cell line constructs expressing specific mutations in ADAR proteins so that we could determine what was required for the interaction between miRNAs precursors and ADARs. We found that these two pathways could antagonise each other however this was very specific and only occurred on certain miRNAs. We also found that this antagonism could be independent of enzymatic activity and that processing of miRNAs could be hindered by binding of the editing enzymes. We obtained a very similar result in Drosophila where an inactive ADAR1 could also inhibited RNA interference via the siRNA pathway.

These findings are significant as they reveal that despite 10-16 % of miRNAs being edited an up-regulation of ADAR1 does not result in additional miRNAs being edited. Instead it appears that editing of miRNA is highly specific and may not have any affect on the functioning of the miRNA. The main affect of RNA editing is on processing of miRNAs with a subsequent reduction in their level. As this does not always require editing activity just binding of the proteins, it is difficult to detect. Therefore if there is a decrease in specific miRNAs levels then their precursors should be analysed to determine if they are substrates of ADARs or bound by them.

The potential impact of these results is on future research on miRNAs. When researchers are investigating a decrease in particular miRNAs it will guide them to determine if ADAR proteins are affecting miRNAs biogenesis even if there is no evidence of RNA editing when the miRNAs are sequenced.

There is no direct economic impact of this research. However it does fulfil the Marie Curie goal of exchange of data and collaboration between groups. As a direct outcome of this project we have established interactions with a group in Berlin and have submitted a manuscript for publication with them. Also Bret Heale is now returning to the USA to take up a Research Assistant Professor position and this Marie Curie fellowship has funded his research and training in the UK.