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Regulation and Function of RNA Editing in the Mammalian Brain
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 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 [en]
RNA editing, ADAR, GABAA receptor, Gabra-3
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-62278ISBN: 978-91-7447-363-6 (print)OAI: oai:DiVA.org:su-62278DiVA: diva2:440803
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
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: 2017-12-12Bibliographically 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
Show others...
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: 2017-12-12Bibliographically 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
Show others...
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: 2017-12-11Bibliographically approved
4. A distant cis acting intronic element induces site-selective RNA editing
Open this publication in new window or tab >>A distant cis acting intronic element induces site-selective RNA editing
Show others...
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-83006 (URN)10.1093/nar/gks691 (DOI)000310377200048 ()
Note

AuthorCount:5;

Available from: 2012-12-05 Created: 2012-12-03 Last updated: 2017-12-07Bibliographically approved

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