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A distant cis acting intronic element induces site-selective RNA editing
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 19, 9876-9886 p.Article in journal (Refereed) Published
Abstract [en]

Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABA(A) receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome.

Place, publisher, year, edition, pages
2012. Vol. 40, no 19, 9876-9886 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-83006DOI: 10.1093/nar/gks691ISI: 000310377200048OAI: oai:DiVA.org:su-83006DiVA: diva2:574521
Note

AuthorCount:5;

Available from: 2012-12-05 Created: 2012-12-03 Last updated: 2017-12-07Bibliographically approved
In thesis
1. A-to-I RNA editing: Function and consequences during brain development
Open this publication in new window or tab >>A-to-I RNA editing: Function and consequences during brain development
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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 GABAA 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. 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2013. 54 p.
Keyword
RNA editing, miRNAs, Brain development
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-93137 (URN)978-91-7447-739-9 (ISBN)
Public defence
2013-10-11, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper 3: Manuscript

Available from: 2013-09-19 Created: 2013-09-03 Last updated: 2013-09-10Bibliographically approved
2. Regulation and Function of RNA Editing in the Mammalian Brain
Open this publication in new window or tab >>Regulation and Function of RNA Editing in the Mammalian Brain
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Adenosine (A) to inosine (I) RNA editing is a widespread post-transcriptional mechanism in eukaryotes that increases the protein diversity. Adenosine deaminases acting on RNA (ADARs) are the enzymes that catalyze this conversion. The diversity generated by ADAR enzymes occurs mainly in the brain where they target transcripts coding for proteins in the central nervous system (CNS).

We have determined the editing frequency of known ADAR substrates during development of the mouse brain using the large-scale 454-sequencing method. We show in paper I that editing is regulated during development of the brain, where it increases along with the maturation of the brain. We propose that the unedited isoform of proteins are required for the undeveloped brain while the edited isoforms are more suitable for the mature brain.

In paper II we show that substrates with multiple editing sites, one specific principle adenosine is favored for initial editing. We demonstrate that within these substrates, editing is coupled when adenosines are located in multiples of twelve nucleotides. These edited adenosines reside on the same side in the tertiary RNA helical structure. A model is suggested where kinetically favored structures at principle editing sites attract ADAR to the substrate, followed by editing at sites that are structurally adjacent to the initiation site.

Editing of the mammalian Gabra-3 transcripts coding for the GABAA receptor α3 subunits recodes an isoleucine (I) to a methionine (M) referred as the I/M site. In paper III we demonstrate that receptors containing edited α3 subunits have altered trafficking properties compared to receptors containing unedited α3 subunits. We suggest that the amino acid residue change, affects protein interactions required for stability and trafficking of GABAA receptors. We propose that the biological function of editing is to reduce the number of α3 subunits in favor of other α subunits.

The dsRNA structure at the I/M site in the Gabra-3 transcript is formed within the exon 9 sequence. We show in paper IV that a conserved intronic dsRNA structure in the downstream intron is required for editing to occur at the I/M site. We demonstrate that in the context of this intronic duplex also non-ADAR substrates can be edited. We propose that the intronic dsRNA stabilize the short I/M stem structure, thereby increasing the ability of ADAR to bind and edit the I/M site. These discoveries have expanded the knowledge in how ADAR editing is employed to supply the development of the brain as well as the RNA structure requirement for editing to occur.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2011. 69 p.
Keyword
RNA editing, ADAR, GABAA receptor, Gabra-3
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-62278 (URN)978-91-7447-363-6 (ISBN)
Public defence
2011-10-21, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2011-09-29 Created: 2011-09-13 Last updated: 2013-09-09Bibliographically approved

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