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Regulation of site-selective A-to-I RNA editing: During mammalian brain development
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University , 2011. , 60 p.
Keyword [en]
RNA editering, ADAR, brain development
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-55525ISBN: 978-91-7447-257-8OAI: oai:DiVA.org:su-55525DiVA: diva2:404896
Public defence
2011-04-15, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (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: 2011-03-24 Created: 2011-03-18 Last updated: 2011-03-18Bibliographically approved
List of papers
1. Large-scale mRNA sequencing determines global regulation of RNA editing during brain development
Open this publication in new window or tab >>Large-scale mRNA sequencing determines global regulation of RNA editing during brain development
2009 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 19, 978-986 p.Article in journal (Refereed) Published
Abstract [en]

RNA editing by adenosine deamination has been shown to generate multiple isoforms of several neural receptors, often with profound effects on receptor function. However, little is known about the regulation of editing activity during development. We have developed a large-scale RNA sequencing protocol to determine adenosine-to-inosine (A-to-I) editing frequencies in the coding region of genes in the mammalian brain. Using the 454 Life Sciences (Roche) Amplicon Sequencing technology, we were able to determine even low levels of editing with high accuracy. The efficiency of editing for 28 different sites was analyzed during the development of the mouse brain from embryogenesis to adulthood. We show that, with few exceptions, the editing efficiency is low during embryogenesis, increasing gradually at different rates up to the adult mouse. The variation in editing gave receptors like HTR2C and GABAA (gamma-aminobutyric acid type A) a different set of protein isoforms during development from those in the adult animal. Furthermore, we show that this regulation of editing activity cannot be explained by an altered expression of the ADAR proteins but, rather, by the presence of a regulatory network that controls the editing activity during development.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-35411 (URN)10.1101/gr.089409.108 (DOI)000266521500003 ()
Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2011-09-29Bibliographically approved
2. Recognition and coupling af A-to-I edited sites are determined by the tertiary structure of the RNA
Open this publication in new window or tab >>Recognition and coupling af A-to-I edited sites are determined by the tertiary structure of the RNA
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2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 20, 6916-6926 p.Article in journal (Refereed) Published
Abstract [en]

Adenosine-to-inosine (A-to-I) editing has been shown to be an important mechanism that increases protein diversity in the brain of organisms from human to fly. The family of ADAR enzymes converts some adenosines of RNA duplexes to inosines through hydrolytic deamination. The adenosine recognition mechanism is still largely unknown. Here, to investigate it, we analyzed a set of selectively edited substrates with a cluster of edited sites. We used a large set of individual transcripts sequenced by the 454 sequencing technique. On average, we analyzed 570 single transcripts per edited region at four different developmental stages from embryogenesis to adulthood. To our knowledge, this is the first time, large-scale sequencing has been used to determine synchronous editing events. We demonstrate that edited sites are only coupled within specific distances from each other. Furthermore, our results show that the coupled sites of editing are positioned on the same side of a helix, indicating that the three-dimensional structure is key in ADAR enzyme substrate recognition. Finally, we propose that editing by the ADAR enzymes is initiated by their attraction to one principal site in the substrate.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-35085 (URN)10.1093/nar/gkp731 (DOI)000271819900024 ()
Available from: 2010-01-14 Created: 2010-01-14 Last updated: 2011-09-29Bibliographically approved
3. Adenosine-to-Inosine RNA Editing Affects Trafficking of the γ-Aminobutyric Acid Type A (GABAA) Receptor
Open this publication in new window or tab >>Adenosine-to-Inosine RNA Editing Affects Trafficking of the γ-Aminobutyric Acid Type A (GABAA) Receptor
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 3, 2031-2040 p.Article in journal (Refereed) Published
Abstract [en]

Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABAA receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABAA receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain.

Keyword
Brain Cell, Surface Receptor, Double-stranded RNA, GABA Receptors, Receptor Modification, RNA Editing, Trafficking
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-52721 (URN)10.1074/jbc.M110.130096 (DOI)000286191500045 ()
Available from: 2011-01-18 Created: 2011-01-18 Last updated: 2012-01-20Bibliographically approved
4. The subcellular localization of ADAR determines A-to-I editing levels in developing neurons
Open this publication in new window or tab >>The subcellular localization of ADAR determines A-to-I editing levels in developing neurons
(English)Manuscript (preprint) (Other academic)
Keyword
RNA editering
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:su:diva-55506 (URN)
Available from: 2011-03-18 Created: 2011-03-18 Last updated: 2011-03-18Bibliographically approved

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