Coordination of editing and splicing of glutamate receptor pre-mRNA

Abstract

Adenosine deaminase that acts on RNA, ADAR, catalyzes the conversion of adenosine into inosine within double-stranded RNA. This type of editing has mainly been found in genes involved in neurotransmission. Site-specific A to I modifications often require intronic sequences to create the double-stranded structure necessary for editing. A system was developed to investigate if editing and splicing of pre-mRNA are coordinated. We have focused on a selectively edited site (R/G) in the glutamate receptor subunit B pre-mRNA. This editing site is situated in close proximity to a 5' splice site. To ensure efficient splicing, the editing site, together with its natural 5' splice site, was fused to a 3' splice site of the major late transcript from adenovirus. In vitro, on a premade transcript, ADAR2 editing and splicing were found to interfere with each other. The stable stem-loop required for ADAR2 editing had a negative effect on in vitro splicing, possibly by sequestering the 5' splice site. Further, RNA helicase A was shown to overcome the splicing inhibition caused by ADAR2. In vivo, allowing cotranscriptional processing, the same construct was found to efficiently edit and splice without interference, suggesting that the two RNA processing events are coordinated.

FIGURE 1.

(A) Schematic representation of a part of rat glutamate receptor subunit B pre-mRNA. Exons are represented by boxes and introns by lines, not drawn to scale. The R/G editing site and editing complementary sequence are shown in detail. Exonic sequences are written in capitals and intronic sequences in lowercase. The sequence deleted in the Δ20 mutant is underlined. (B) The editing/splicing reporter constructs used in the study. All constructs contain exon 13 including the R/G editing site and the 5′ part of intron 13. GRG 988 contains full-length intron 13 and exon 14, whereas GRG SI has a shortened intron. GRG SS contains part of an intron, the 3′ splice site and exon from a modified adeno major late transcript.

FIGURE 2.

Efficient splicing and editing of the GRG SS pre-mRNA. (A) In vitro splicing of GRG SS pre-mRNA and GRG SS pre-mRNA lacking the ECS (Δ20). Splicing products were separated on denaturing polyacrylamide gel. The position of nonspliced pre-mRNA and spliced mRNA is indicated to the right of the gel; exons are represented by boxes and introns by lines. The percentage of splicing is indicated below the gel. (B) Diagram of in vitro splicing efficiency of GRG SS wild type and Δ20 pre-mRNA. Splicing efficiency was plotted against time. Data represents three independent experiments, error bars show standard error of the mean, SEM. (C) GRG SI and GRG SS pre-mRNAs can be edited in vitro by recombinant rADAR2a (A2) as shown by limited primer extension. Position of primer and products corresponding to nonedited, R/G, and -1 edited pre-mRNAs are indicated. Numbers below show the amount of editing in percentage in the different substrates.

FIGURE 3.

(A) ADAR2 inhibited splicing of GRG SS pre-mRNA. In vitro splicing of GRG SS or GRG SS Δ20 pre-mRNA with or without recombinant rADAR2a (A2) was analyzed using RT-PCR at time points as indicated. Products were separated on a nondenaturing 6% polyacrylamide gel. Additional upper bands have been sequenced and correspond to full-length pre-mRNA. The position of precursor and spliced mRNA are indicated on the right, exons are represented by boxes, and introns by lines. Used for size determination was 1 kb PLUS marker (Life Technologies™). The percentage of splicing is indicated below the gel. (B) Splicing and editing are competing in vitro. In vitro editing/splicing reactions with preincubation of either nuclear extract (NE) or recombinant rADAR2a (A2) for 5 min, as indicated. PCR products corresponding to pre-mRNA and mRNA at 30 min of splicing reaction were sequenced, and the chromatograms with the R/G editing site indicated by an arrow are shown. ATP was omitted (-ATP) in the splicing reaction in the middle panel. (C) RNA helicase A (RHA) enhanced in vitro splicing of GRG SS and counteracted splicing inhibition by ADAR2 (A2). In vitro editing/splicing reactions with preincubation of recombinant rADAR2a and/or RHA, as indicated. Two controls are included: a splicing reaction without nuclear extract (No NE), and a control splicing reaction (C) containing an equal volume of ADAR storage buffer without rADAR2a. The positions of precursor and spliced mRNA are indicated to the right of the gel; exons are represented by boxes and introns by lines. Used for size determination was 1 kb PLUS marker (Life Technologies™). The percentage of splicing is indicated below the gel.

FIGURE 4.

(A) ADAR2 edits reporter constructs at the R/G site in vivo. Transfection of 293 cells with editing/splicing reporter constructs with or without ADAR2 expression vector. Total RNA was isolated and RT-PCR products corresponding to either spliced or nonspliced RNA were sequenced. The top left sequence corresponds to a nonedited R/G site, indicated by an arrow. Cotransfection with ADAR2 expression vector results in editing seen as a mixed A and G peak at the R/G editing site in the top right and bottom chromatograms. (B) GRG SI and 988 pre-mRNAs were inefficiently spliced in vivo, whereas GRG SS RNA was only detectable as spliced mRNA. The degree of splicing was not affected by coexpression of ADAR2. RT-PCR products corresponding to pre-mRNA and spliced mRNA were separated on 2% agarose gel. Used for size determination was 1 kb PLUS marker (M) (Life Technologies™).

FIGURE 5.

Model of editing and splicing of GluR-B pre-mRNA in vitro and in vivo. (A) ADAR2 (black circle) competes with splicing factors (SF, gray circle) for binding to the R/G stem-loop in vitro. Disruption of the stem-loop by RNA helicase A (RHA, checked oval) facilitates the binding of splicing factors and thereby enhances splicing. (B) In vivo, editing and splicing is coordinated, possibly by the CTD (black rectangle) of RNA pol II. RHA might be the link between RNA processing factors and the polymerase. Editing (1.) and splicing (2.) are sequential events occurring in vivo.