The aim of my thesis has been to study how A-to-I RNA editing of miRNAs is regulated during brain development and the biological function of these editing events.

Using high throughput RNA sequencing, we performed an unbiased search for edited, mature miRNAs in total mouse brain tissue from three developmental stages. We searched for known and novel editing sites within short RNA sequences approximately resembling the length of mature miRNAs.

We can conclude that the gradual increase in editing efficiency seen for most selectively edited sites in transcripts encoding neurotransmission proteins, also applies to miRNAs during development of the mammalian brain. The most striking editing events all occur in the crucial seed sequence, essential for target recognition. These results indicate that A-to-I editing is utilized to diversify target recognition by the miRNAs during development.

Furthermore, our data suggests that specific transcripts, targeted by either non-edited or edited miRNAs, are regulated in a manner that is consistent with the developmental shifts in editing frequencies. One example of this is the developmentally regulated editing of miR-381, targeting the Pum2 transcript in the brain. Pum2 is a translational repressor that regulates many mRNAs shown to be important for neurological functions, including memory formation and learning.

We have further analyzed what determines a substrate to be edited by the ADAR enzymes, specifically in the context of the mammalian GABA_A receptor. We found that long stem loop structures located close to exon sequences function as inducers of exonic editing.

Taken together, my research demonstrate the power of combining, RNA-Seq, bioinformatics and specific experimental verifications in order to shed light on the impact of A-to-I editing on the process of RNA interference. Furthermore, we have expanded the knowledge of RNA structure requirements for ADAR editing to occur. 

The transcriptome of the mammalian brain is extensively modified by adenosine to inosine (A-to-I) nucleotide conversion by two adenosine deaminases (ADAR1 and ADAR2). As adenosine and inosine have different base pairing properties, A-to-I RNA editing shapes the functional output of both coding and non-coding RNAs (ncRNAs) in the brain. The aim of this thesis was to identify editing events in small regulatory ncRNAs (miRNAs) and to determine their temporal and spatial editing status in the developing and adult mouse brain. To do this, we initially analyzed the editing status of miRNAs from different developmental time points of the mouse brain. We detected novel miRNA substrates subjected to A-to-I editing and found a general increase in miRNA editing during brain development, implicating a more stringent control of miRNAs as the brain matures. Most of the edited miRNAs were found to be transcribed as a single long consecutive transcript from a large gene cluster. However, maturation from this primary miRNA (pri-miRNA) transcript into functional forms of miRNAs is regulated individually, and might be influenced by the ADAR proteins in an editing independent matter. We also found that edited miRNAs were highly expressed at the synapse, implicating a role as local regulators of synaptic translation. We further show that the increase in editing during development is explained by a gradual accumulation of the ADAR enzymes in the nucleus. Specifically for ADAR2, we found a developmentally increasing interaction with two factors, importin-α4 and Pin1, that facilitate nuclear localization of the editing enzyme. We have also found that selectively edited stem loops often are flanked by other long stem loop structures that induce editing in cis. This may explain why multiple pri-miRNAs are edited within the same cluster. In conclusion, this thesis has significantly increased the understanding of the dynamics of both editing substrates and enzymes in the developing and mature brain.