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. 2006 Mar;7(3):303-7.
doi: 10.1038/sj.embor.7400621. Epub 2006 Jan 27.

RNA editing and alternative splicing: the importance of co-transcriptional coordination

Jurga Laurencikiene  1 , Annika M Källman, Nova Fong, David L Bentley, Marie Ohman
Affiliations

RNA editing and alternative splicing: the importance of co-transcriptional coordination

Jurga Laurencikiene et al. EMBO Rep. 2006 Mar.

Abstract

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (pol II) is essential for several co-transcriptional pre-messenger RNA processing events, including capping, 3'-end processing and splicing. We investigated the role of the CTD of RNA pol II in the coordination of A to I editing and splicing of the ADAR2 (ADAR: adenosine deaminases that act on RNA) pre-mRNA. The auto-editing of Adar2 intron 4 by the ADAR2 adenosine deaminase is tightly coupled to splicing, as the modification of the dinucleotide AA to AI creates a new 3' splice site. Unlike other introns, the CTD is not required for efficient splicing of intron 4 at either the normal 3' splice site or the alternative site created by editing. However, the CTD is required for efficient co-transcriptional auto-editing of ADAR2 intron 4. Our results implicate the CTD in site-selective RNA editing by ADAR2 and in coordination of editing with alternative splicing.

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Figures

Figure 1
Figure 1
Rat Adar2 Ex 4–5 minigene. Predicted secondary structure of intron 4 of Adar2. For technical reasons, the stem has been shortened with approximately 40 base pairs interrupted by bulges and loops. A to I editing that leads to alternative splicing is in red. Other sites of editing are in blue. Normal and alternative acceptor splice sites are underlined.
Figure 2
Figure 2
ADAR2 pre-mRNA processing and the influence of the carboxy-terminal domain. (A) A2 Ex 4–5 (lanes 2,3) and A2 Ex 4–5 G (lanes 4,5) were analysed by reverse transcription–PCR (RT–PCR). Expected band size for alternative splicing was 387 base pairs (bp) and for normal splicing 340 bp. Alternative (alt.) and normal (norm.) splicing is indicated on the right. The size of fragments in lane 1 is in base pairs. (B) Western blot analysis of exogenously expressed ADAR2 in human embryonic kidney 293 cells without (−) and with (+) a transiently transfected ADAR2 expression vector. An α-actin antibody is used as a control for equal loading. (C) Editing/splicing analysed by RT–PCR. The A2 Ex4–5 reporter was transcribed by a wild-type RNA polymerase II (pol II; WT, lanes 2,3) or a pol II with C-terminal domain deletion (ΔCTD, lanes 4,5). The presence or absence of ADAR2 expression is indicated by (+) or (−). Alternative (alt.) and normal (norm.) splicing is indicated on the right. (D) Average percentage of alternative splicing of the A2 Ex 4–5 RNA in the presence of ADAR2 for transfected wild-type (WT) and CTD-deleted pol II (ΔCTD) and three independent experiments transcribed by endogenous pol II (endo). (E) Western blot analysis of exogenous rADAR2 in cells transfected with α-amanitin-resistant polymerases. The level of ADAR2 was normalized to the expression of actin as indicated on the right. The size of proteins indicated to the left in the marker lane (MW) is in kilodaltons.
Figure 3
Figure 3
Splicing and alternative splicing of a ‘pre-edited' ADAR2 substrate. (A) Spliced A2 Ex4–5 G RNA transcribed by a wild-type RNA polymerase II (pol II; lane 2) or pol II ΔCTD (lane 3). Alternative (alt.) and normal (norm.) splicing is indicated on the right. The size of fragments in lane 1 is in base pairs. (B) Average percentage of alternative splicing of the A2 Ex 4–5 G RNA was from four independent experiments.
Figure 4
Figure 4
Editing and alternative splicing of transcripts using different carboxy-terminal domain deletion mutants. (A) Alternative (alt.) and normal (norm.) splicing was analysed by reverse transcription–PCR (RT–PCR). The A2 Ex4–5 reporter was transcribed by an RNA polymerase II (pol II) with a wild-type C-terminal domain (CTD; WT), CTD deleted (ΔCTD), heptad repeats 1–25 with the additional ten-residue C-terminal motif (1–25+Cterm.) and repeats 27–52 including the ten-residue C-terminal motif (27–52+Cterm.). The size of fragments in lane 1 is in base pairs. Bottom: semiquantitative RT–PCR with 32P-labelled primers shows expression of ADAR2 in each lane. (B) Quantification of RT–PCR results using the different CTD deletion mutants. The A2 Ex4–5 reporter was transcribed by an RNA pol II with a wild-type CTD (WT), a deleted CTD (ΔCTD), heptad repeats 1–25 with the additional ten-residue C-terminal motif (1–25+C) or heptad repeats 27–52 including the ten-residue C-terminal motif (27–52+C). The average percentage of alternatively spliced PCR products from two independent experiments is shown.
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Comment in

  • An editor controlled by transcription.
    Riedmann EM, Jantsch MF. Riedmann EM, et al. EMBO Rep. 2006 Mar;7(3):269-70. doi: 10.1038/sj.embor.7400650. EMBO Rep. 2006. PMID: 16607395 Free PMC article. No abstract available.

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