Controlling the A-to-I editing landscape

Adenosine to Inosine RNA-editing (A-to-I editing) is a co-transcriptional process where a genomically encoded adenosine is deaminated and converted into inosine. Editing can have diverse consequences, for instance the deaminated adenosine may cause a codon change and thus one amino acid in the encoded protein can be different. Editing is a highly dynamic process and editing levels range from below 1% to almost 100%. Thus, it has been speculated that editing can help to fine-tune the transcript, which may help to adjust the cell to stress situations. De-regulation of editing levels has been linked to various diseases including cancer, depression, or epilepsy. Thus, it is important for the cell to tightly control editing levels. Moreover, editing levels also seem to be important in the light of development as they generally increase from embryonic to mature stages. Finally, editing levels also differ between tissues. Surprisingly, we found very recently that the editing levels even differ dramatically between pre-mRNA and the mature transcript. Here, we plan to unravel the underlying factors that cause the observed differences. These may be either explained by differences in stability, selective export, or selective processing of edited or unedited RNA. We will address these hypotheses systematically. Firstly, we plan to carefully determine editing levels for a panel of conserved protein-coding editing targets for different stages of transcript maturation in order to see where editing levels change. Secondly, we will determine stability of edited RNA. Thirdly and most importantly, we will use model substrates (edited or unedited) and incubate them with cellular extracts. Using specific purification techniques and mass spectrometry this will reveal factors differentially binding to edited or unedited RNAs. Lastly, for validation we propose to deplete cells from the identified factors and check if the depletion indeed changes the ratio between pre-mRNA editing levels and editing levels of the mature transcript. The proposed work is highly significant as factors controlling editing levels are largely unknown. To our knowledge nothing is known about the control of editing levels during transcript maturation. Thus, we break new ground when we aim at identifying regulatory factors, which may help to understand the differences in editing levels between tissues, during development, or even de-regulation of editing during disease.

Adenosine deamination is a frequent post-transcriptional modification in RNAs. Deamination of adenosines leads to the formation of inosines. Inosines are frequently interpreted as guanosines by most cellular machineries. Consequently, adenosine deamination can alter the coding potential of RNAs and of genetic information. In this project we aimed at deciphering factors that control deamination of adenosines in RNAs. We could demonstrate an intimate connection between adenosine deamination and RNA-splicing and an impact of both processes on each other. Most interestingly, we could also demonstrate that transcriptional dynamics has a major impact on RNA-editing. The mechanisms underlying this phenomenon are currently unknown. In a separate study we could show that inosines are not only interpreted as guanosines but also as adenosines and uracils during translation.