Differential recruitment of pre-mRNA splicing factors to alternatively spliced transcripts in vivo

Abstract

Alternative splicing in mammalian cells has been suggested to be largely controlled by combinatorial binding of basal splicing factors to pre-mRNA templates. This model predicts that distinct sets of pre-mRNA splicing factors are associated with alternatively spliced transcripts. However, no experimental evidence for differential recruitment of splicing factors to transcripts with distinct splicing fates is available. Here we have used quantitative single-cell imaging to test this key prediction in vivo. We show that distinct combinations of splicing factors are recruited to sites of alternatively spliced transcripts in intact cells. While a subset of serine/arginine protein splicing factors, including SF2/ASF, SC35, and SRp20, is efficiently recruited to the tau gene when exon 10 is included, these factors are less frequently associated with tau transcription sites when exon 10 is excluded. In contrast, the frequency of recruitment of several other splicing factors is independent of splicing outcome. Mutation analysis of SF2/ASF shows that both protein-protein as well as protein-RNA interactions are required for differential recruitment. The differential behavior of the various splicing factors provides the basis for combinatorial occupancy at pre-mRNAs. These observations represent the first in vivo evidence for differential association of pre-mRNA splicing factors with alternatively spliced transcripts. They confirm a key prediction of a stochastic model of alternative splicing, in which distinct combinatorial sets of generic pre-mRNA splicing factors contribute to splicing outcome.

Figure 1. Characterization of Stable Cell Lines Expressing Alternatively Spliced tau Minigenes

(A) Schematic representation of the tau exon 10 minigene system. Alternative inclusion or exclusion of exon 10 as observed for endogenous tau is recapitulated in a minigene containing flanking HIV-TAT exons. (B) Preferential inclusion of tau exon 10 detected by gel electrophoresis. The wttau exon 10 (tau10wt) is evenly included or excluded from the mRNA, whereas a tau exon 10 containing a T>C mutation at position −1 of exon 10 (tau10−1) is predominantly included in the same context. Exon 10 inclusion is detected as a 246-bp by RT-PCR using primers (arrows) in the TAT exons. Exon 10 exclusion is detected by a 153-bp band. (C) Quantitative real-time PCR analysis to determine exon 10 inclusion. Results are averages ± standard deviations from at least three experiments. (D) RNA-FISH using nick-translated probes against the tau minigene to detect sites of tau transcription. The vast majority of cells in a stable cell population contained one or two tau minigene insertion sites. Arrowheads indicate tau transcription sites. Scale bar = 3.5 μm.

Figure 2. Differential Recruitment of Endogenous Pre-RNA Splicing Factors to Alternatively Spliced Transcripts

(A) Recruitment of splicing factors to tau transcription sites detected by combined RNA-FISH using specific probes against the tau minigene (green) and IF microscopy with specific anti-splicing factor antibodies (red). Arrowheads indicate tau transcription sites. All associations were confirmed by linescan analysis. Scale bar = 2.5 μm. (Inset: higher magnifications of the transcription site) Lines indicate the location of the linescans. (B) Quantitation of percentage of cells with colocalization of tau RNA-FISH and splicing factor signals. Values represent averages from at least 100 transcription sites from at least three experiments ± SEM.

Figure 3. Recruitment of Endogenous Pre-RNA Splicing Factors to Transcription Sites Containing tau Exon 10 mRNA

(A) Recruitment of splicing factors to tau transcription sites detected by combined RNA-FISH using a specific probe against tau exon 10 (green) and IF microscopy with specific anti-splicing factor antibodies (red). All associations were confirmed by linescan analysis. Scale bar = 2.5 μm. (B) Quantitation of percentage of cells with colocalization of tau RNA-FISH and splicing factor signals. Values represent averages from at least 100 transcription sites from at least three experiments ± SEM. Recruitment to the subpopulation of transcription sites containing predominantly tau exon10 mRNA in tau10wt cells was as efficient as recruitment in tau10−1 cells.

Figure 4. Mutation-Independent Differential Recruitment of Endogenous Pre-RNA Splicing Factors

(A) Stable cell lines expressing minigenes containing a G>A mutation at +3 (tau10+3) or a C>U mutation at +14 (tau10+14). Both mutations give preferential inclusion of exon 10 as previously reported for transient expression. (B) Quantitative real-time PCR analysis demonstrating the preferential inclusion of exon 10 in tau10+3 and tau10+14 compared to tau10wt. Values represent averages ± standard deviations from at least three experiments. Values of included exon 10 are normalized to total minigene RNA. (C) Quantitative analysis of recruitment of endogenous splicing factors to tau10+3 or tau10+14. Recruitment of splicing factors to tau transcription sites detected by combined RNA-FISH using specific probes against the tau minigene and IF microscopy with specific anti-splicing factor antibodies. Percentage of cells with colocalization of tau RNA-FISH and splicing factor signals is indicated. Values represent averages from at least 100 transcription sites from three experiments ± SEM.

Figure 5. Differential Recruitment of Exogenous Pre-RNA Splicing Factors to tau Minigenes

(A) Semi-quantitative RT-PCR analysis of tau alternative splicing upon expression of T7-tagged SR proteins in COS-7 cells expressing either tau10wt or tau10−1. Transient transfection of splicing factors does not affect the alternative splicing pattern of the tau minigene in stable cell lines. (B) Quantitative analysis of recruitment of transfected SR proteins to tau10wt or tau10−1. Recruitment of splicing factors to tau transcription sites detected by combined RNA-FISH using specific probes against the tau minigene and IF microscopy with anti-T7 antibody. Percentage of cells with colocalization of tau RNA-FISH and splicing factor signals is indicated. Values represent averages from at least 100 transcription sites from three experiments ± SEM.

Figure 6. Deletion Mapping of Sf2/Asf Protein Domains Involved in Differential Recruitment

(A) Schematic representation of T7-tagged mutants of SF2/ASF. (B) Semi-quantitative RT-PCR analysis of tau10−1 splicing upon expression of mutant SF2/ASF. Overexpression of the mutant proteins does not affect tau splicing. (C) Recruitment of splicing factors to tau transcription sites detected by combined RNA-FISH using specific probes against the tau minigene (green) and IF microscopy with anti-T7 antibody (red). Arrowheads indicate tau transcription sites. Scale bar = 2.5 μm. (D) Quantitation of percentage of cells with colocalization of tau RNA-FISH and splicing factor signals. Values represent averages from at least 50 transcription sites from three experiments ± SEM.