Caspase-2 pre-mRNA alternative splicing: Identification of an intronic element containing a decoy 3' acceptor site

Abstract

We have established a model system using the caspase-2 pre-mRNA and initiated a study on the role of alternative splicing in regulation of programmed cell death. A caspase-2 minigene construct has been made that can be alternatively spliced in transfected cells and in nuclear extracts. Using this system, we have identified a 100-nt region in downstream intron 9 that inhibits the inclusion of the 61-bp alternative exon. This element (In100) can facilitate exon skipping in the context of competing 3' or 5' splice sites, but not in single-intron splicing units. The In100 element is also active in certain heterologous pre-mRNAs, although in a highly context-dependent manner. Interestingly, we found that In100 contains a sequence that highly resembles a bona fide 3' splice site. We provide evidence that this sequence acts as a "decoy" acceptor site that engages in U2 snRNP-dependent but nonproductive splicing complexes with the 5' splice site of exon 9, hence conferring competitive advantage to the exon-skipping splicing event (E8-E10). These results reveal a mechanism of action for a negative intronic regulatory element and uncover a role for U2 snRNP in the regulation of alternative splicing.

Figure 1

An intronic element inhibits casp-2 exon 9 inclusion. (A) Structure of casp-2 deletion mutant constructs. The gray box in the intron indicates the location of the In100 element. The small cross-hatched rectangle depicts “filler” sequences used as a size control. Positions of oligonucleotides used for RT-PCR are indicated. (B) RT-PCR analysis was carried out using total RNA extracted from cells transfected with the constructs shown in A. Ratio of casp-2_S to casp-2_L (%) was determined from five independent experiments and plotted in the histogram below. (C) ³²P-labeled transcripts were incubated in HeLa cell nuclear extracts under standard splicing conditions. Splicing products were resolved on an 8% acrylamide/8 M urea gel. The black and white arrowheads indicate the position of the casp-2_L (skipping) and casp-2_S (inclusion) mRNAs, respectively.

Figure 2

In100 requires the context of competing splice sites. (A) Structure of splicing substrates. The thinner line and shaded rectangle represent β-globin sequences substituted in the C2/C3Gloi2 substrates. The gray box in parentheses depicts the presence or the absence of the In100 element. The table shows the splicing efficiency (%) as determined from three independent experiments. (B) Splicing of Ex9–10 substrates. Substrates were incubated in HeLa nuclear extracts for the time (in hours) indicated above each lanes. (C) Splicing of Ex8–10 substrates. (D) Splicing of 553 derivatives. Position of the splicing products generated from the use of the proximal (Prox.) or distal (Dist.) splice site is indicated by the white or black triangles, respectively. (E) Splicing of the (C2/C3)Gloi2 derivatives in transfected HeLa cells. Position of exon 9 inclusion and skipping mRNAs is shown.

Figure 3

U1 snRNP occupancy of casp-2 competing 5′ splice sites as determined by an RNase H protection assay. (A) Substrates containing (C2–553) or lacking (C3–553) In100 were tested in a U1 snRNP protection assay. The protected and cleaved RNA species were resolved on an 8% polyacrylamide denaturing gel as indicated. Data from five independent experiments are represented below in the histogram. U1 snRNP-dependent complexes responsible for the observed protection are depicted by a white sphere on each 5′ splice site. (B) The U1 snRNP RNase H protection assay was carried out as shown in A except that substrates lacking exon 10 and its associated 3′ splice site were used. As a control, the protection was also done with HeLa nuclear extracts depleted of functional U1 snRNP (U1⁻; lanes 1 and 5). (Histogram) Relative levels of protection at the proximal 5′ splice site were plotted over time for each substrate with the data from two independent experiments. (C) In100 contains a sequence resembling a 3′ acceptor site. Putative branch site (BS) and polypyrimidine track [(Py)n] are shown. The arrow above the sequence shows the potential cleavage site at the 3′ splice site AG. (D) The upstream portion of In100 can be used as a functional 3′ splice site. Standard splicing reactions were set up using substrates containing exon 9 and its 5′ donor site with downstream intron sequences terminated immediately downstream of In100 (i9In100) or control substrates as depicted in Fig. 5A.

Figure 4

In100 acts as a decoy 3′ splice site. (A) A model for function of In100 as a decoy 3′ splice site. White spheres stand for U1 snRNP-dependent 5′ splice site complexes, and gray spheres depict U2 snRNP-dependent complexes forming on In100 or on the native 3′ splice site of exon 10. Relative levels of U1 snRNP-dependent protection are represented by + signs below each 5′ splice site. Relative use of distal and proximal 5′ splice sites are indicated on the right. In this model, the 5′ splice site of alternative exon 9 engages in nonproductive splicing complexes with the decoy 3′ splice site in the In100 element, as shown by the dotted line between arrow heads. This in turn confers a competitive advantage to the exon 9-skipping splicing pathway, as shown by the long solid line between the arrow heads. (B) Minimal fragments containing the wild-type or the mutated 3′ acceptor site region of In100 were inserted back into the C3 construct (sequence of In50up fragments is indicated). Mutated nucleotides are indicated with small dots under the sequence. These constructs were transfected into HeLa cells, and the splicing profile was analyzed by RT-PCR as before. The numbers below the gels show the percentage of exon 9 inclusion as quantified using a PhosphorImager. (C) In50up and In50upΔ fragments were inserted in the C3–553 substrate (In50up- and In50upΔ-553 substrates, respectively), and the U1 snRNP RNase H protection assay was carried out as before. Positions of proximal and distal 5′ splice site protected fragments are as indicated.

Figure 5

Analysis of complexes assembled on In100. (A) Gel electrophoresis analysis of splicing complexes formed on the substrates shown above the gels. Heterogeneous (H) and spliceosomal complexes (A–C) are indicated on the left. (B) U2AF⁶⁵ interacts with the In100 element. UV cross-linking was performed following incubation of labeled RNA substrates with 30, 60, or 120 ng of purified U2AF⁶⁵; 120 ng of U2AF⁶⁵ protein was used for i9 and C3i9. Cross-linked proteins were resolved on 12.5% SDS/PAGE. (C) Nuclear factors specifically interact with In100. Labeled RNA substrates containing the In100 or the native 3′ splice site of exon 10 were subjected to UV cross-linking following incubation with purified U2AF⁶⁵ or HeLa nuclear extracts (lanes 1 and 2 or lanes 3 and 4, respectively). In100 cross-linking reactions were subjected to immunoprecipitation with a monoclonal antibody directed against U2AF⁶⁵ (lane 7) or FCS (lane 6). The black arrowhead and asterisk indicate positions of specific 65- and 35-kDa cross-linking proteins, as described in the text.