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. 2009 Jun;19(6):978-86.
doi: 10.1101/gr.089409.108. Epub 2009 May 6.

Large-scale mRNA sequencing determines global regulation of RNA editing during brain development

Helene Wahlstedt  1 , Chammiran Daniel, Mats Ensterö, Marie Ohman
Affiliations

Large-scale mRNA sequencing determines global regulation of RNA editing during brain development

Helene Wahlstedt et al. Genome Res. 2009 Jun.

Abstract

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 GABA(A) (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.

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Figures

Figure 1.
Figure 1.
Editing levels of the glutamate receptor transcripts at four different developmental stages; E15, E19, P2, and P21 of the mouse brain determined by 454 Amplicon Sequencing after RT-PCR. (A) The percentage of transcripts edited at Q/R site, the nearby +4 site, and the R/G site in Gria2. (B) Editing frequency at the R/G position of the Gria3 transcripts. (C) The level of editing at the Q/R site of the Grik1 transcripts. (D) Editing frequency at the Q/R, Y/C and I/V sites of the Grik2 transcripts.
Figure 2.
Figure 2.
Efficiency of editing in the transcripts of Gabra3, Cyfip2, Flna, and Kcna1 at four developmental stages, E15, E19, P2, and P21 of the mouse brain determined by 454 Amplicon Sequencing. (A) Editing frequency at the I/M site of Gabra3 transcripts. (B) Editing frequency at the K/E site of Cyfip2 transcripts. (C) Editing frequency at the Q/R site of Flna transcripts. (D) Editing levels at the I/V site of the Kcna1 transcripts.
Figure 3.
Figure 3.
Editing of the serotonin receptor at the four developmental stages E15, E19, P2, and P21. (A) Editing levels at the A, B, C′, C, and D sites of the Htr2c transcripts was determined by 454 Amplicon Sequencing after RT-PCR. (B) The distribution of the HTR2C protein isoforms during development as a consequence of the editing events.
Figure 4.
Figure 4.
Editing of the Adarb1 transcripts during development and the ratio between normal and alternatively spliced Adarb1 transcripts. (A) Editing frequency at the −1, 10, 23, and 24 sites of Adarb1 transcripts was determined by 454 Amplicon Sequencing of RT-PCR products from four developmental stages, E15, E19, P2, and P21. (B) Detection of normal and alternatively spliced Adarb1 transcripts by RT-PCR during the six developmental stages, E15, E19, P2, P14, P21, and >P21. (C) Protein levels of ADAR and ADARB1 during development of the mouse brain. Western blot analysis showing the ADAR (110 kDa) and ADARB1 expression during the developmental stages, E15, E19, P2, P14, and P21 as indicated. An anti-actin antibody was used as a loading control.
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