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Recognition and coupling af A-to-I edited sites are determined by the tertiary structure of the RNA
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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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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.

Place, publisher, year, edition, pages
2009. Vol. 37, no 20, 6916-6926 p.
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-35085DOI: 10.1093/nar/gkp731ISI: 000271819900024OAI: oai:DiVA.org:su-35085DiVA: diva2:286357
Available from: 2010-01-14 Created: 2010-01-14 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Regulation of site-selective A-to-I RNA editing: During mammalian brain development
Open this publication in new window or tab >>Regulation of site-selective A-to-I RNA editing: During mammalian brain development
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
RNA editering, ADAR, brain development
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-55525 (URN)978-91-7447-257-8 (ISBN)
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
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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