The impact of adenosine-deamination type RNA editing on pre-mRNA splicing

In eukaryotes, genetic information stored in DNA in the nucleus is transcribed into RNA which is transported to the cytoplasm where it is translated into proteins. This flow of information can be changed by two important post-transcriptional processes, alternative pre- mRNA splicing and RNA-editing. Both processes can alter the genetic information leading to the translation of proteins that differ from the genomically encoded information therefore leading to a regulated but transient diversification of genetic information. In metazoa, nucleotide changes introduced by adenosine deaminases that act on RNA (ADARs) are the most abundant type of RNA-editing that can affect thousands of mRNAs in mammals. Similarly, alternative splicing is also most abundant in mammals. To allow the formation of novel functional mRNAs both processes need to be regulated and coordinated in a spatio-temporal order. Interestingly, both processes, RNA-editing by ADARs and alternative splicing occur in the nucleus, and are most likely cotranscriptionally coupled. Studies on isolated substrates have shown that editing can create splice sites, influence the rate of splicing, and influence splice site choice. Here we propose to study the impact of ADAR-mediated RNA editing on pre-mRNA splicing. This will be done by a transcriptome-wide analysis of alternative splice patterns in mouse tissues impaired in ADAR-mediated RNA editing. Technically, this challenging task will be accomplished by combining two novel RNA sequencing methods. To gain mechanistic insight, the global approach will be complemented by a detailed study on model substrates where editing influences splicing. Here the folding state, the assembly of splicing factors, and the binding of splice regulators will be compared in the absence and presence of editing. Thus, our study will give insight how the two most important processes leading to post transcriptional changes in genetic information are coupled, both quantitatively and mechanistically.

In this research project we aimed at understanding the interplay of two RNA maturation processes, pre-mRNA splicing and RNA-editing. We could show that both processes are intimately interconnected and can strongly influence each other. Genetic information is stored in DNA and transmitted from one cell to its daughter cells. For genetic information to be used it needs to be transcribed into RNA. Certain RNAs exhibit cellular functions themselves while other RNAs need to be translated into proteins to exert their functions. More than 30 years of research have shown that RNA is not only a mere copy of the genetic information but can also be modified and altered, leading to diversification of the genetic information. Important mechanisms of RNA-diversification are alternative splicing and RNA-editing. During alternative splicing, regions of the RNA are removed and the remaining parts are reassembled. Depending on the regions removed, genetic information can be slightly modified this way. During RNA-editing the identity of individual bases is changed by deamination reactions. This way, cytidine can be converted to uridine and adenine can be converted to inosine. As uridin is interpreted as thymin and inosine as guanine during translation, novel proteins can be formed that are not encoded in the genome. Both, pre-mRNA splicing and RNA-editing take place in the nucleus, likely before the RNA is completely transcribed. In the course of the project we investigated how the absence of two RNA-editing enzymes ADAR1 and ADAR affect pre-mRNA splicing. We could show that editing has a massive impact on pre-mRNA splicing. Most interestingly, the absence of ADAR1 or ADAR2 affects splicing in different ways suggesting that the underlying mechanisms by which these two enzymes affect splicing will most likely differ.