A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2

Abstract

Splicing of mouse immunoglobulin (IgM) exons M1 and M2 is directed by two juxtaposed regulatory elements, an enhancer and an inhibitor, located within the M2 exon. A primary function of the enhancer is to counteract the inhibitor, allowing splicing to occur. Here we show that the inhibitor contains two binding sites for polypyrimidine tract binding protein (PTB). Mutational analysis indicates that only one of these sites is necessary and sufficient to direct splicing inhibition both in vitro and in vivo. We demonstrate that the difference in activity of the two sites is explained by proximity to the intron. We further show that the presence of the enhancer results in the disruption of the PTB-inhibitor interaction, enabling splicing to occur. In the absence of the enhancer, splicing can be artificially activated by immuno-inhibition of PTB. Collectively, our results indicate that a single PTB binding site can function as an inhibitor that regulates alternative splicing both in vitro and in vivo.

FIGURE 1.

Binding of PTB to the IgM M2 splicing inhibitor and disruption by the enhancer. (A) Schematic diagrams of the IgM1–2 pre-mRNA substrate and deletion derivatives lacking the enhancer (IgMΔE) or containing only the inhibitor (IgM-I). M1 and M2, exons M1 and M2, respectively; E, enhancer; INH, inhibitor; I and II, PTB binding sites I and II, respectively. (B) PTB was analyzed for its ability to bind IgM1–2, IgMΔE, IgM-I or a non-specific RNA control (NS RNA) using a UV cross-linking assay. HeLa nuclear extract was incubated with a uniformly ³²P-labeled pre-mRNA substrate for 30 or 60 min in the presence or absence of ATP as indicated. (C) Splicing complexes formed on IgMΔE and IgM1–2 were fractionated on a nondenaturing gel (left), and analyzed for PTB binding using the UV cross-linking assay (right). The PTB-pre-mRNA interaction was analyzed in the inhibitor complex (complex I), prespliceosome (complex A), mature spliceosome (complex B, C), or nonspecific H complex.

FIGURE 2.

Binding of PTB mediates splicing inhibition on IgM1–2 pre-mRNA. (A) In vitro splicing of the IgMΔE pre-mRNA substrate in nuclear extract (NE) following addition of an anti-PTB antibody, or addition of recombinant PTB to the immuno-inhibited extract. (B) In vitro splicing of the IgM-MS2 pre-mRNA substrate in nuclear extract following addition of an MS2-RS domain fusion-protein, MS2 alone, or MS2 plus the anti-PTB antibody. (C) In vitro splicing of the IgM-MS2 pre-mRNA substrate in S100 extract following addition of an MS2-RS domain fusion-protein, the serine-arginine protein ASF, or the anti-PTB antibody, as indicated. Schematic diagrams of the IgM1–2, IgMΔE and IgM-MS2 pre-mRNA substrates are shown.

FIGURE 3.

A single PTB binding site mediates splicing inhibition in vitro. (A) Mutant derivatives of the IgM-I RNA, in which either or both of the PTB binding sites were mutated, were analyzed for their ability to bind PTB in vitro using an ultraviolet (UV) cross-linking assay. (B) PTB binding site mutant derivatives of the IgMΔE pre-mRNA substrate were analyzed using an in vitro splicing assay. The sequence of the IgM M2 inhibitor showing the putative PTB binding sites and the mutant versions is shown. (C) Adenovirus major late (Ad ML) pre-mRNA derivatives containing either site I or site II inserted into exon 2 were analyzed using an in vitro splicing assay. (D) An IgMΔE derivative in which site I was mutated and site II was replaced with the site I sequence (IgMΔE-sub1) was analyzed using an in vitro splicing assay. The sequence of the inhibitor in the wild type and IgMΔE-sub1 pre-mRNA substrates is shown.

FIGURE 4.

A single PTB binding protein binding site mediates splicing inhibition in vivo. IgM1–2, IgMΔE, and mutant derivatives of IgMΔE in which either or both of the PTB binding sites were mutated, were analyzed using an assay that monitored splicing of pre-mRNA substrates in transfected COS7 cells. (A) (Top) RNA was analyzed by RT-PCR using two sets of primer-pairs: IgM1–5′ and IgM1–2SJ, which overlaps the splicing junction between the first and second exons, were used to detect spliced products; IgM1–5′ and IgM1–3′ were used to detect both spliced and unspliced RNAs. As a control, a reaction was also performed in the absence of reverse transcriptase (RT–). (Bottom) PCR bands were quantitated, and the ratio of spliced product to the total RNA (spliced and unspliced) was calculated and normalized to the amount of spliced IgM1–2 product. (B) RNA was analyzed by RT-PCR using the IgM1–5′ /IgM2 primer-pair to investigate additional cryptic splicing products. Schematic diagrams of the IgM1–2 and IgME substrates and the location of the primers are shown.