Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5' or 3' splice site activation - PubMed (original) (raw)

Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5' or 3' splice site activation

C F Bourgeois et al. Mol Cell Biol. 1999 Nov.

Abstract

The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5' splice sites and of one major or one minor 3' splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5' splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5' splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3' splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5' splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

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Figures

FIG. 1

FIG. 1

A purine-rich sequence separately activates two distinct splicing reactions. (A) Structure of the Sp4 transcript and mRNA for alternative splicing reactions of the adenovirus type 2 E1A unit. Boxes represent the exons, and lines represent the introns. Note that sequences represented by hatched boxes and grey boxes act as intronic sequences when 9S or 12S splicing reactions occur. The hairpin structure of the 216-nt intron is indicated (12). Restriction sites: B, _Bst_XI; D, _Dra_III; X, _Xma_I. Different mRNA isoforms obtained by alternative splicing of the Sp4 transcript are schematized below the pre-mRNA structure. (B) Sequence of the 12S 5′ss region of the E1A unit. Nucleotides 936 to 978 (in the adenovirus type 2 viral genome) of the wild-type (wt) sequence are represented. Exonic nucleotides are in uppercase, and intronic nucleotides are in lowercase. The BSE is underlined and divided into two halves, namely, Pu1 and Pu2. Only the mutated nucleotides of the different constructs are indicated. (C) In vitro splicing assays. Splicing reactions were carried out with various transcripts, as indicated above the lanes. Lanes 1 to 8, transcripts with wild-type splice sites; lanes 9 and 10, transcripts with mutant 12S 5′ss; lanes 11 to 16, transcripts with mutant 13S 5′ss. Products of the different splicing reactions are indicated along the sides of the gels. Asterisks, mRNA obtained by using two cryptic 5′ss located between the 12S and the 13S 5′ss (55); arrowhead, a premature transcription termination.

FIG. 2

FIG. 2

The BSE binds several SR proteins specifically. (A) UV cross-linking experiments were done with [α-32P]ATP-labeled wild-type (wt) or various mutated BSE RNAs (see Fig. 1B for sequences) in the presence of the S100 fraction (lane 1), S100 supplemented by a total-SR preparation (lane 2), or NE (lanes 3 to 8). (B) Immunoprecipitation assay with [α-32P]CTP-labeled wild-type BSE. Standard UV cross-linking reaction mixtures with NE were immunoprecipitated with antibodies specific for individual SR proteins as indicated at the top of each lane (9G8, ASF/SF2, or SC35). The amounts of immunoprecipitated loaded samples correspond to about four times the amounts of control samples. (C) Immunoprecipitation assays with [α-32P]ATP-labeled probes. Experimental conditions were as described for panel B, with BSE and derivatives used as probes, as indicated above each gel. Only 9G8 and ASF/SF2 antibodies were used in this experiment. The time exposure for lanes 5 and 6 corresponds to three times that for lane 4.

FIG. 3

FIG. 3

SR proteins activate 12S and 216-nt IVS splicing reactions differentially. (A) In vitro splicing of transcripts with wild-type (wt) splice sites. Complementation of the splicing of two different transcripts (wt Sp4 [lanes 1 to 4] and Pu2 x2 transcript [lanes 5 to 8]) was performed in limiting conditions (lanes 1 and 5, assays without SR protein), as described in Materials and Methods, or with fixed amounts of individual purified recombinant SR proteins 9G8, ASF/SF2, and SC35, as indicated at the top of each lane. (B) Same experiment as that shown in panel A, but with mutant 13S 5′ss transcripts. Lanes 1 and 5, assays without supplemented SR protein.

FIG. 4

FIG. 4

9G8 factor activates 12S splicing specifically in a BSE-dependent manner in a heterologous context. (A) Schematic representation of the transcript with duplicated 12S 5′ss. Two copies of the BSE-lacking 12S 5′ss (D) each surrounded by 4 exonic nucleotides (open region at base of arrowhead) and by 36 intronic nucleotides (thick line) were inserted between _Bam_HI (B) and _Xba_I (X) sites of a linker at the place of the natural E1A 13S 5′ss, to give rise to the D/D construct. Light grey boxes and thin line: natural exonic and intronic regions of the E1A unit, respectively. The proximal site was next replaced by a wild-type (D) or an improved (D+) 12S 5′ss, preceded by the wild-type or mutant BSE (mBSE), giving rise to four new constructs: D/BSE-D, D/mBSE-D, D/BSE-D+, and D/mBSE-D+. (B) In vitro splicing assays. Standard experiments using NE (lanes 1 to 6 and 11) or complementation experiments with a fixed amount of SR proteins (lanes 7 to 10 and 12 to 15) were performed as described in Materials and Methods. Splicing products resulting from the utilization of the proximal (prox.), distal (dist.), or cryptic (crypt.) 5′ss (located 88 nt upstream of the distal site) are indicated alongside the gel. Lanes 7 and 12, assays without SR protein.

FIG. 5

FIG. 5

The BSE exhibits the same effect in vivo as in vitro. (A) Representation of the minigene used in transfection experiments. The E1A unit (see also Fig. 1 legend) was subcloned in the pXJ41 vector (80) under the control of the human cytomegalovirus promoter, followed by simian virus 40 (SV-40) polyadenylation signals. Primers used for RT and PCR are represented by arrows. The position of the BSE is indicated. (B) Results of RT-PCR. Wild-type (wt) E1A or mutants mBSE, mPu1, and mPu2 (see Fig. 1B for sequences) were transfected in HeLa cells (see Materials and Methods for details). Total RNA was extracted and analyzed by RT-PCR followed by nondenaturing polyacrylamide gel electrophoresis. Lanes 2 and 11, PCR control with pXJ41-E1A plasmid as a matrix; lane 1, pSP65/MspI marker (501, 489, 404, 242, 228, 223 and 190 bp). Different sets of primers were used according to the various splicing reactions analyzed. In each panel, the downstream primer was used for both RT and PCR. Primers a and c, general E1A splicing pattern; primers b and d, 13S and 12S mRNA analysis; primers a and e, 13S and 11S mRNA analysis.

FIG. 6

FIG. 6

Model for alternative effect of SR proteins on E1A 12S and 216-nt IVS splicing reactions through the BSE. (A) General activation mechanism of both splicing reactions by the BSE. Only the region from the 216-nt IVS 3′ss to the 12S 5′ss is schematized, with lines representing the introns and boxes representing the exon. Sequences of Pu1 and Pu2 motifs of the BSE are indicated under the exon. Both halves of the BSE are necessary to activate efficiently the upstream 216-nt IVS splicing reaction, whereas only the Pu1 motif is required for 12S reaction activation. (B) Activation hypothesis for the downstream 12S splicing reaction. 9G8 binds to the first part of the BSE (Pu1 motif) and stabilizes the binding of the U1 snRNP at the 5′ splice site, either directly by interaction with one component of the U1 snRNP or indirectly. (C) Hypotheses for the coordinated activation of the upstream splicing reaction. ASF/SF2, SC35, or possibly SRp40 proteins bind to the BSE and enhance the recognition of the weak 216-nt IVS 3′ss, likely by protein-protein interactions with U2AF35. We do not know if the 13S 5′ss-mediated activation involves direct interactions with the splicing machinery at the 216-nt IVS 3′ss, as a simple exon definition process, or if interactions with the BSE activation complex occur first.

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