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Selective ADAR editing and the coordination with splicing
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Maturation of precursor messenger RNAs (pre-mRNA) in eukaryotes includes processes like capping, 3’ end formation and splicing. Some premRNAs undergo an additional process called RNA editing where a single nucleotide is modified to generate a new nucleotide identity. One such RNA editing event is the hydrolytic deamination of adenosine (A) that results in inosine (I). Because of its base pairing properties, inosine is recognized as guanosine (G) by cellular machineries, like the spliceosome or the ribosome. The enzymes catalyzing A to I editing are the ADARs (Adenosine deaminases that act on RNA). In vivo, these enzymes selectively locate and deaminate individual adenosines within a few substrates, all with long double stranded helixes interrupted by bulges and internal loops. It is not yet fully understood how ADARs discriminate between sites for selective editing and other adenosines in a double stranded context. Edited sites are often located near exon/intron borders where editing depends on intron sequences to form the double stranded structure required for ADAR recognition. Thus editing has to occur prior to splicing. In the work described in this thesis we have investigated the mechanism of selective editing and the possible coordination of editing and splicing.

We have found that ADAR1 and ADAR2 display different editing specificities for a natural substrate, in vitro. ADAR2, but not ADAR1 perform site selective editing, similar to what occurs in vivo. Further, we have found that the selective editing is not determined by mismatches in the vicinity of the edited site. It is rather the immediate structure surrounding the adenosine that affects editing selectivity by ADAR2 in vitro.

The coordination of editing and splicing was investigated in vivo. We found that the C-terminal domain (CTD) of the RNA polymerase II is required for efficient ADAR2 editing during ongoing transcription, while splicing of substrates for editing is independent of the CTD.

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik , 2004. , 52 p.
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-302ISBN: 91-7265-982-3 (print)OAI: oai:DiVA.org:su-302DiVA: diva2:192194
Public defence
2004-12-10, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 13:00
Opponent
Supervisors
Available from: 2004-11-22 Created: 2004-11-22Bibliographically approved
List of papers
1. In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site
Open this publication in new window or tab >>In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site
2000 In: RNA, ISSN 1355-8382, Vol. 6, 687-697 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23460 (URN)
Note
Part of urn:nbn:se:su:diva-302Available from: 2004-11-22 Created: 2004-11-22Bibliographically approved
2. ADAR2 A to I editing: site selectivity and editing efficiency are separate events
Open this publication in new window or tab >>ADAR2 A to I editing: site selectivity and editing efficiency are separate events
2003 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 16, 4874-4881 p.Article in journal (Refereed) Published
Abstract [en]

ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double‐stranded. Site‐selective A→I editing has been detected at specific sites within  a few tructured pre‐mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre‐mRNA of the glutamate receptor subunit B (GluR‐B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double‐stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.                 

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-23461 (URN)10.1093/nar/gkg681 (DOI)
Available from: 2004-11-22 Created: 2004-11-22 Last updated: 2017-12-13Bibliographically approved
3. Biochemical analysis and scanning force microscopy reveal productive and non-productive ADAR2 binding to RNA substrates
Open this publication in new window or tab >>Biochemical analysis and scanning force microscopy reveal productive and non-productive ADAR2 binding to RNA substrates
Show others...
2003 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 9, no 7, 839-846 p.Article in journal (Refereed) Published
Abstract [en]

Scanning force microscopy (SFM) can be used to image biomolecules at high resolution. Here we demonstrate that single-molecule analysis by SFM complements biochemical data on RNA protein binding and can provide information that cannot be obtained by the usual biochemical methods. We have used this method to study the interaction between the RNA editing enzyme ADAR2 and RNA transcripts containing selective and nonselective editing sites. The natural selectively edited R/G site from glutamate receptor subunit B (GluR-B) was inserted into an RNA backbone molecule consisting of a completely double-stranded (ds) central part and incompletely paired ends derived from potato spindle tuber viroid (PSTVd). This molecule was efficiently edited at the R/G site, but promiscuous editing occurred at nonselective sites in the completely double-stranded region. The construct was also used to analyze binding of ADAR2 to wild-type and modified R/G editing sites in relation to binding at other nonselectively edited sites. Editing analysis together with SFM allow us to differentiate between binding and enzymatic activity. ADAR2 has                     been reported to have a general affinity to dsRNA. However, we show that there is a prominent bias for stable binding at sites selectively edited over other edited sites. On the other hand, promiscuous editing at nonselective sites apparently results from transient binding of the enzyme to the substrate. Furthermore, we find distinct sites with nonproductive binding of the enzyme.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-23462 (URN)10.1261/rna.2167603 (DOI)
Available from: 2004-11-22 Created: 2004-11-22 Last updated: 2017-12-13Bibliographically approved
4. RNA editing and alternative splicing: the importance of co-transcriptional coordination
Open this publication in new window or tab >>RNA editing and alternative splicing: the importance of co-transcriptional coordination
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23463 (URN)
Note
Part of urn:nbn:se:su:diva-302Available from: 2004-11-22 Created: 2004-11-22 Last updated: 2010-01-13Bibliographically approved

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