First come, first served revisited: factors affecting the same alternative splicing event have different effects on the relative rates of intron removal

Abstract

Alternative splicing accounts for much of the complexity in higher eukaryotes. Thus, its regulation must allow for flexibility without hampering either its specificity or its fidelity. The mechanisms involved in alternative splicing regulation, especially those acting through coupling with transcription, have not been deeply studied in in vivo models. Much of our knowledge comes from in vitro approaches, where conditions can be precisely controlled at the expense of losing several levels of regulation present in intact cells. Here we studied the relative order of removal of the introns flanking a model alternative cassette exon. We show that there is a preferential removal of the intron downstream from the cassette exon before the upstream intron has been removed. Most importantly, both cis-acting mutations and trans-acting factors that regulate the model alternative splicing event differentially affect the relative order of removal. However, reduction of transcriptional elongation causing higher inclusion of the cassette exon does not change the order of intron removal, suggesting that the assumption, according to the "first come, first served" model, that slow elongation promotes preferential excision of the upstream intron has to be revised. We propose instead that slow elongation favors commitment to exon inclusion during spliceosome assembly. Our results reveal that measuring the order of intron removal may be a straightforward read-out to discriminate among different mechanisms of alternative splice site selection.

FIGURE 1.

Fibronectin E33 (EDI or EDA) is spliced through a pathway that removes its downstream intron prior to the upstream one. (A) Diagrams of the two possible pathways leading to E33 inclusion and the two resulting splicing intermediates. Thin rectangles depict weak (gray) and strong (black) 3′ splice sites. Arrows depict the primers used to detect and quantify the proximal and distal intermediates, hereafter named α and β, respectively. (B) Hep3B cells transfected with the reporter minigene pUHC-EDA were analyzed by RT-PCR for E33 inclusion ratios of both the minigene and the endogenous E33 using specific primers (left, gels; center, quantification, gray bars). (Right) Real-time RT-PCR analysis for the quantification of splicing intermediates calculated as the %α-intermediate (black bars) as explained in Materials and Methods. (C) Real-time RT-PCR of proximal (a,b) and distal (c,d) pre-mRNA regions of Hep3B cells transfected with a ΔDRE mutant of the reporter minigene pUHC-EDA. Proximal region: Primer b (5′-GCATTCAGACACCCAAGAAC-3′) was used for cDNA synthesis; primers a (5′-TTCTCTGCACAGCTCCTAAG-3′) and b were used for PCR. Distal region: Primer e (5′-GGTATTTGGAGG TCAGCA-3′) was used for cDNA synthesis; primers c (5′-TTGGAACTACGTTTATTTTCC-3′) and d (5′-GCGGCCAGGGGTCACGAT-3′) were used for PCR as previously described (de la Mata et al. 2003). A.U. indicates arbitrary units. (D) Quantification of the α intermediates (empty circles) and β intermediates (black squares) of Hep3B cells transfected with pUCH-EDA at different times after treatment with actinomycin D. A.U. indicates arbitrary units. Calculations show mean ± SD.

FIGURE 2.

Different cis-acting mutations enhance E33 inclusion through different intron removal pathways. (A) Diagrams representing the two basic minigenes used in this study. (B) Mutations introduced in the minigenes shown in A. WT indicates wild-type sequence; GT and DM, G→T and G→T+A→T point mutations, respectively, at the polypyrimidine tract of the 3′ss upstream of E33 (Nogues et al. 2003); ΔDRE, deletion of the intronic downstream regulatory element (Gromak et al. 2008). The minigenes containing two alternative splicing regions, WT (pFN-EDI^WT/IIICS) and DM (pFN-EDI^C/IIICS), were previously described (Fededa et al. 2005). (C,D) Hep3B cells were transfected with the WT and mutant versions of pUHC-EDA (C) and pFN-EDI/IIICS (D), and analyzed by RT-PCR for E33 inclusion ratios (left), the quantification of E33+/E33− ratios (middle), and the quantification of splicing intermediates (right, black bars) calculated as the %α-intermediate. Calculations show mean ± SD.

FIGURE 3.

SR proteins regulate E33 inclusion through different order of intron removal pathways. Hep3B cells were cotransfected with the reporter minigene pUHC-EDA and either vectors expressing different SR proteins or SR protein-specific siRNAs. (A) Radioactive RT-PCR analysis of E33 isoforms and quantification of E33+/E33− ratios (gray bars). (B) Real-time RT-PCR analysis for the quantification of splicing intermediates calculated as the %α-intermediate (black bars). Calculations show mean ± SD.

FIGURE 4.

Inhibition of pol II elongation does not affect the relative rates of intron removal. Hep3B cells were transfected with the reporter minigene pUHC-EDA and treated with the ethanol vehicle (lane 1) or 25 μM DRB for 8 h (lane 2) following induction by withdrawal of tetracycline. (Lanes 3,4) Cells were cotransfected in the presence of tetracycline with the reporter minigene pUHC-EDA and expression vectors for human α-amanitin resistant pol II large subunits (hRpb1), WT (lane 3) and slow mutant (C4, lane 4). Minigene transcription was induced 18 h post-transfection by washing out tet and adding α-amanitin (10 μg mL⁻¹) to inhibit endogenous pol II transcription. Cells were harvested 20 h post-induction and used for subsequent assays. (A) Results of radioactive RT-PCR analysis of E33 isoforms and quantification of E33+/E33− ratios (gray bars). (B) Real-time RT-PCR analysis for the quantification of splicing intermediates calculated as %α-intermediate (black bars). Calculations show mean ± SD.

FIGURE 5.

Alternative models for the “first come, first served” mechanism of splice site selection. (A) Fast elongation promotes usage of the stronger downstream 3′ splice site. (B) Slow elongation causes preferential excision of the upstream intron (first served equals first excised). (C) Slow elongation causes commitment to E33 inclusion via recruitment of splicing factors (first served equals first committed). Both introns are excised individually and in an order that is independent of elongation.