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Regulation of RNA Editing: The impact of inosine on the neuronal transcriptome
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholms universitet. (Marie Öhman)ORCID iD: 0000-0002-3272-1377
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The transcriptome of the mammalian brain is extensively modified by adenosine to inosine (A-to-I) nucleotide conversion by two adenosine deaminases (ADAR1 and ADAR2). As adenosine and inosine have different base pairing properties, A-to-I RNA editing shapes the functional output of both coding and non-coding RNAs (ncRNAs) in the brain. The aim of this thesis was to identify editing events in small regulatory ncRNAs (miRNAs) and to determine their temporal and spatial editing status in the developing and adult mouse brain. To do this, we initially analyzed the editing status of miRNAs from different developmental time points of the mouse brain. We detected novel miRNA substrates subjected to A-to-I editing and found a general increase in miRNA editing during brain development, implicating a more stringent control of miRNAs as the brain matures. Most of the edited miRNAs were found to be transcribed as a single long consecutive transcript from a large gene cluster. However, maturation from this primary miRNA (pri-miRNA) transcript into functional forms of miRNAs is regulated individually, and might be influenced by the ADAR proteins in an editing independent matter. We also found that edited miRNAs were highly expressed at the synapse, implicating a role as local regulators of synaptic translation. We further show that the increase in editing during development is explained by a gradual accumulation of the ADAR enzymes in the nucleus. Specifically for ADAR2, we found a developmentally increasing interaction with two factors, importin-α4 and Pin1, that facilitate nuclear localization of the editing enzyme. We have also found that selectively edited stem loops often are flanked by other long stem loop structures that induce editing in cis. This may explain why multiple pri-miRNAs are edited within the same cluster. In conclusion, this thesis has significantly increased the understanding of the dynamics of both editing substrates and enzymes in the developing and mature brain.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2017. , 51 p.
Keyword [en]
RNA editing, ADAR, miRNA, Neuron, Brain development, Synapse
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-142324ISBN: 978-91-7649-729-6 (print)ISBN: 978-91-7649-730-2 (electronic)OAI: oai:DiVA.org:su-142324DiVA: diva2:1092049
Public defence
2017-06-09, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrheniusväg 20, 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 2: Manuscript.

Available from: 2017-05-17 Created: 2017-04-29 Last updated: 2017-05-15Bibliographically approved
List of papers
1. A-to-I editing of microRNAs in the mammalian brain increases during development
Open this publication in new window or tab >>A-to-I editing of microRNAs in the mammalian brain increases during development
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2012 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 22, no 8, 1477-1487 p.Article in journal (Refereed) Published
Abstract [en]

Adenosine-to-inosine (A-to-I) RNA editing targets double-stranded RNA stem-loop structures in the mammalian brain. It has previously been shown that miRNAs are substrates for A-to-I editing. For the first time, we show that for several definitions of edited miRNA, the level of editing increases with development, thereby indicating a regulatory role for editing during brain maturation. We use high-throughput RNA sequencing to determine editing levels in mature miRNA, from the mouse transcriptome, and compare these with the levels of editing in pri-miRNA. We show that increased editing during development gradually changes the proportions of the two miR-376a isoforms, which previously have been shown to have different targets. Several other miRNAs that also are edited in the seed sequence show an increased level of editing through development. By comparing editing of pri-miRNA with editing and expression of the corresponding mature miRNA, we also show an editing-induced developmental regulation of miRNA expression. Taken together, our results imply that RNA editing influences the miRNA repertoire during brain maturation.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-81732 (URN)10.1101/gr.131912.111 (DOI)000307090300010 ()
Note

AuthorCount:5;

Available from: 2012-10-30 Created: 2012-10-30 Last updated: 2017-05-08Bibliographically approved
2. Synaptic expression and regulation of miRNA editing in the brain
Open this publication in new window or tab >>Synaptic expression and regulation of miRNA editing in the brain
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In the brain, sophisticated networks of RNA regulatory events tightly control gene expression in order to achieve proper brain function. We and others have previously shown that several miRNAs, encoded within the miR-379-410 cluster, are subjected to A-to-I RNA editing. In the present study we conclude these edited miRNAs to be transcribed as a single long consecutive transcript, however the maturation into functional forms of miRNAs is regulated individually. In seven of the miRNAs, subjected to editing, we analyze how editing relates to miRNA maturation. Of particular interest has been maturation of miR-381-3p and miR-376b-3p, both important for neuronal plasticity, dendrite outgrowth and neuronal homeostasis. Most of the edited miRNAs from the cluster, are highly edited in their unprocessed primary transcript, including miR-381-3p and miR-376b-3p. However, editing in miR-381-3p is almost entirely absent in the mature form, while editing is increased in the mature form of miR-376b-3p compared to the primary transcript. We propose that ADAR1 positively influences the maturation of pri-miR-381 in an editing independent manner. In pri-miR-376b we hypothesize that ADAR1 and ADAR2 competes for editing, and while ADAR2 inhibits miRNA maturation, ADAR1 editing is frequently present in the mature miR-376b-3p. We further show that miR-381-3p and miR-376b-3p regulate the dendritically expressed Pumilio 2 (Pum2) protein. By next generation RNA sequencing (NGS RNA-seq) on purified synaptoneurosomes, we show that miR-381-3p is highly expressed at the synapse, suggesting its functional role in locally regulating Pum2. Furthermore, we identify a set of highly expressed miRNAs at the synapse, which may act locally to target synaptic mRNAs.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-142315 (URN)
Available from: 2017-04-28 Created: 2017-04-28 Last updated: 2017-05-08Bibliographically approved
3. Accumulation of nuclear ADAR2 regulates A-to-I RNA editing during neuronal development
Open this publication in new window or tab >>Accumulation of nuclear ADAR2 regulates A-to-I RNA editing during neuronal development
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2017 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 130, 745-753 p.Article in journal (Refereed) Published
Abstract [en]

Adenosine to inosine (A-to-I) RNA editing is important for a functional brain, and most known sites that are subject to selective RNA editing have been found to result in diversified protein isoforms that are involved in neurotransmission. In the absence of the active editing enzymes ADAR1 or ADAR2 (also known as ADAR and ADARB1, respectively), mice fail to survive until adulthood. Nuclear A-to-I editing of neuronal transcripts is regulated during brain development, with low levels of editing in the embryo and a dramatic increase after birth. Yet, little is known about the mechanisms that regulate editing during development. Here, we demonstrate lower levels of ADAR2 in the nucleus of immature neurons than in mature neurons. We show that importin-a4 (encoded by Kpna3), which increases during neuronal maturation, interacts with ADAR2 and contributes to the editing efficiency by bringing it into the nucleus. Moreover, we detect an increased number of interactions between ADAR2 and the nuclear isomerase Pin1 as neurons mature, which contribute to ADAR2 protein stability. Together, these findings explain how the nuclear editing of substrates that are important for neuronal function can increase as the brain develops. 

Keyword
A-to-I RNA editing, ADAR2, Importin-alpha-4, Pin1
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-142128 (URN)10.1242/jcs.200055 (DOI)000394456200010 ()
Available from: 2017-04-25 Created: 2017-04-25 Last updated: 2017-05-31Bibliographically approved
4. Alu elements shape the primate transcriptome by cis-regulation of RNA editing
Open this publication in new window or tab >>Alu elements shape the primate transcriptome by cis-regulation of RNA editing
2014 (English)In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 15, no 2, R28Article in journal (Refereed) Published
Abstract [en]

Background: RNA editing by adenosine to inosine deamination is a widespread phenomenon, particularly frequent in the human transcriptome, largely due to the presence of inverted Alu repeats and their ability to form double-stranded structures - a requisite for ADAR editing. While several hundred thousand editing sites have been identified within these primate-specific repeats, the function of Alu-editing has yet to be elucidated. Results: We show that inverted Alu repeats, expressed in the primate brain, can induce site-selective editing in cis on sites located several hundred nucleotides from the Alu elements. Furthermore, a computational analysis, based on available RNA-seq data, finds that site-selective editing occurs significantly closer to edited Alu elements than expected. These targets are poorly edited upon deletion of the editing inducers, as well as in homologous transcripts from organisms lacking Alus. Sequences surrounding sites near edited Alus in UTRs, have been subjected to a lesser extent of evolutionary selection than those far from edited Alus, indicating that their editing generally depends on cis-acting Alus. Interestingly, we find an enrichment of primate-specific editing within encoded sequence or the UTRs of zinc finger-containing transcription factors. Conclusions: We propose a model whereby primate-specific editing is induced by adjacent Alu elements that function as recruitment elements for the ADAR editing enzymes. The enrichment of site-selective editing with potentially functional consequences on the expression of transcription factors indicates that editing contributes more profoundly to the transcriptomic regulation and repertoire in primates than previously thought.

National Category
Genetics Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-105262 (URN)10.1186/gb-2014-15-2-r28 (DOI)000336256600011 ()
Funder
Swedish Research Council, K2013-66X-20702-06-4
Note

AuthorCount:4;

Available from: 2014-06-25 Created: 2014-06-24 Last updated: 2017-05-08Bibliographically approved

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