Mice with altered RNA-editing patterns

RNA editing by ADARs converts adenosines to inosines in double-stranded and structured RNAs. Inosines are interpreted as guanosines by most cellular processes and therefore A to I conversion can have profound impact on the coding potential, splicing, folding, and localization of an RNA, just to name a few. In mice, ADAR-mediated RNA-editing is essential for normal life and development. Consistently, hyper and hypo-editing have been found associated with several diseases, predominantly of the nervous system. The detailed molecular, cellular, and organismic consequences of RNA-editing are, however, only known in a few instances. RNA-editing is very abundant in repetitive elements located in non-coding regions. The large number of editing events in these regions also precludes their individual analysis at the organismic level. RNA editing in coding RNAs, in contrast, is less abundant. Here, editing can affect the coding potential of an RNA, leading to the formation of a protein that differs from the genomically encoded version. Recently, we and others identified several highly conserved editing events in protein coding genes. Here we propose to study the impact of editing on the function of three of these proteins at the cellular and organismic level. The three proteins chosen, filamin alpha, filamin beta, and cyFIP2 have well documented functions and are all involved in the organization of the actin cytoskeleton. We will therefore compare the cellular localization, influence on actin polymerization, and intracellular interactions of edited and constitutively unedited versions of these proteins. The main focus of this project, however, will be the generation and analysis of transgenic mice in which editing in two of these genes has been specifically abolished. To this end, a transgenic mouse, unable to edit the filamin-alpha pre-mRNA has been generated. In summary, our study will shed light on the impact of RNA-editing on the function and organization of the actin cytoskeleton.

Genetic information stored in DNA is typically copied into RNA before being translated into proteins. This central dogma of molecular biology is broken by the process of RNA-editing, where genetic information is changed after it has been copied into an mRNA. In metazoa, adenosine deamination by adenosine deamination acting on RNA (ADAR) enzymes is the most abundant form of RNA editing. Millions of editing sites have been identified in mammalian mRNAs, mostly located in the 3 UTRs of mRNAs but also found in their coding regions. Adenosine deamination leads to the formation of Inosines, which have the basepairing potential of guanosines. Hence, adenosine to Inosine conversion in mRNAs can alter codons and lead to the translation of a protein that differs from the genomically encoded version.The mRNA encoding the actin crosslinking protein Filamin A (FLNA) is found edited from birds to mammals. Editing at position 7022 of the mouse and human open reading frame leads to a Q to R amino acid exchange at position 2341 in the encoded protein. As this editing event is highly conserved we aimed at understanding the physiological consequences of this editing event. To achieve this, we generated mice, exclusively impaired in FLNA mRNA editing.Comparing wild type and transgenic mice that are unable to edit the FLNA pre-mRNA we have shown that editing is highest in tissues that contain smooth muscle cells. Further analysis has shown that smooth muscle cells that lack FLNA editing have an increased density in actin bundles. Consequently, smooth muscle cells lacking FLNA editing hypercontract and mice lacking FLNA editing have increased aortic tension and elevated blood pressure.High editing levels are also observed in the large intestine of mice. We have therefore tested for abnormalities in gastrointestinal function of mice deficient in FLNA editing. Most interestingly, these mice are hypersensitive to inflammatory bowel disease. The molecular mechanisms underlying this phenomenon are currently being investigated.