Regulation of site-selective A-to-I RNA editing During mammalian brain development

Sammanfattning: Adenosine (A) to inosine (I) RNA editing is a widespread post-transcriptional mechanism in mammals that contributes to increase the protein diversity. Adenosine deaminases that act on RNA (ADARs) are the enzymes catalyzing RNA editing. ADARs are particularly active within the brain where they act on transcripts involved in neurotransmission. In this work the editing efficiency of all known site-selectively edited substrates have been analyzed during development of the mouse brain. We show that there is a global regulation of RNA editing, where editing levels of sites increase as the brain matures. This increase in editing efficiency cannot be explained by an increase in ADAR protein expression. During differentiation of primary cells from the mouse brain, editing levels increases similar to what we observe in vivo. Interestingly, the subcellular localization of the ADAR enzymes of cultured neurons show a different distribution in immature compared mature neurons. An accumulation of the ADAR enzymes in the nucleus may explain elevated A-to-I editing during brain development. Furthermore, we find that certain adenosines work as principal sites where editing of the transcript is initiated. Presumably, these sites are kinetically favored and are hypothesized to recruit the ADAR enzymes to the RNA substrate. Editing is then coupled to sites located in multiples of 12 nucleotides from each other. Interestingly, these sites reside on the same side in the 3D helix structure. The Gabra-3 transcript is site-selectively edited at a single position changing an isoleucine codon for a methionine upon editing. Gabra-3 encodes the a3 subunit of the GABAA receptor. We show that receptors assembled with edited a3 are less stable at the cell surface than the non-edited a3. We propose that the amino acid change upon editing, could affect protein interactions important for trafficking and stability of the GABAA receptors. Further, the editing event in a3 may have the function to reduce the number of a3 subunits in favor of other a subunits.