SR proteins induce alternative exon skipping through their activities on the flanking constitutive exons

Abstract

SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.

FIG. 1.

CaMKIIδ alternative splicing in response to SR protein depletion in MEFs. (A) Alternative splicing of CaMKIIδ and detection of its major isoforms by splice junction-specific primers. Constitutive exons are shown as white boxes, and alternative exons are shown as gray boxes. The function of the alternative exons in mediating differential intracellular targeting of the kinase is also indicted. (B) Induction of SRSF2 and SRSF1 depletion in conditional knockout MEFs. (C) Analysis of CaMKIIδ isoforms in wild-type or SRSF2- or SRSF1-deficient MEFs by RT-PCR. The number of PCR cycles is indicated in parentheses for each experiment. (D) Quantification of the induced CaMKIIδ isoform containing exon 16 (E16) by quantitative RT-PCR. Because of the noise during the detection of the CaMKIIδ isoform containing both exons 15 and 16 (E15/16) by real-time PCR, quantification of the isoform was based on the specific band on the gel, which was first normalized against glyceradehyde-3-phosphate dehydrogenase (GAPDH) and then against the major skipped isoform (E13 to E17). Error bars are based on three independent experiments. Statistical significance was determined by a two-tailed t test, and the significant changes are labeled.

FIG. 2.

Exon strength analysis on a splicing reporter. (A) Identification of putative ESEs in the alternative and flanking constitutive exons of the CaMKIIδ gene based on the ESEfinder program. (B) The β-globin minigene (DUP4CX)-based reporter. Individual CaMKIIδ exons (exons 13 through 17) are flanked by the identical intronic splicing signals. Arrows indicate the primers for RT-PCR analysis. (C) Wild-type MEF lines were transfected by the chimeric β-globin minigenes, and the inclusion of each CaMKIIδ exon was analyzed by RT-PCR. (D) Binding levels of individual wt and mutant exons and an intron control with SRSF2 or SRSF1 were measured by UV cross-linking using MEF-derived nuclear extracts containing HA-tagged SR proteins. (E) The inclusion of individual CaMKIIδ exons induced by SR protein depletion.

FIG. 3.

Mutational analysis of CaMKIIδ alternative splicing. (A and B) The CaMKIIδ-based minigenes (CAM16 and CAM15/16) and their splicing patterns in transfected wt and conditional knockout MEFs before and after induced depletion of the specific SR proteins indicated. (C) Induction of exon 16 inclusion by overexpression of individual SR proteins. (D) Mutations of ESEs in exons 16 and 17. Arrows show the ESE scores on the mutated regions. (E and F) RT-PCR analysis of the wt and mutant CaMKIIδ minigenes in transfected MEFs. The results are quantified at the bottom in each panel. Error bars are based on three independent experiments.

FIG. 4.

Induction of exon skipping or inclusion by a tethered RS domain. (A) DUP4-16-based constructs. The CaMKIIδ exon 16-containing splicing reporter was used to individually engineer wt and mutant MS2 binding sites in each exon. The expected sizes of pre-mRNA and spliced mRNA are indicated on the right. (B) The parental reporter and individual MS2-containing constructs labeled on the top were transfected into wt MEFs with either MS2 or the MS2-RS fusion gene as indicated at the bottom. The gel shows the results of RT-PCR analysis. (C) Quantification of the results in panel B. Error bars are based on three independent experiments, and statistical significance was determined by a two-tailed t test.

FIG. 5.

Induction of CaMKIIδ alternative splicing by ectopic expression of tissue-specific splicing regulators and their synergy with SR protein depletion. (A) Effects of ectopic expression of tissue-specific splicing regulators on CaMKIIδ alternative splicing. The plasmids encoding each tissue-specific splicing regulator were transfected into wild-type MEFs, and alternative splicing of the endogenous CaMKIIδ gene was analyzed by RT-PCR. HA-tagged RBFOX proteins and endogenous β-actin were quantified by Western blotting. (B) The putative RBFOX binding sites in the CaMKIIδ minigene and the mutations introduced to the binding sites. (C) RBFOX binding site-mediated splicing response of the CAM15/16 minigene. Individual wild-type and mutant minigene reporters were cotransfected with pcDNA3 (−) or with a plasmid (+) expressing the mouse RBFOX1 gene. Quantified results are shown by the bar graph on the bottom. (D) Synergy between SR protein depletion and overexpression of tissue-specific alternative splicing regulators in the regulation of CaMKIIδ alternative splicing. Individual RBFOX expression plasmids were transfected into wild-type or SRSF2- or SRSF1-deficient MEFs, followed by RT-PCR analysis of the CaMKIIδ isoforms expressed from the CAM15/16 minigene. Quantified results are shown by the bar graph on the bottom, error bars are based on three independent experiments, and statistical significance was determined by a two-tailed t test.

FIG. 6.

Effects of SRSF1 overexpression on the CaMKIIδ splicing program during RA-induced neuronal differentiation. (A) RA treatment of parental P19 cells. (B) RA treatment of SRSF1-overexpressing P19 cells. The time course is indicated on the top, and the expression of individual genes was monitored by semiquantitative RT-PCR, with the specific number of PCR cycles in parentheses in each case. Individual CaMKIIδ isoforms were detected with splice junction-specific primers. (C) RT-PCR analysis of the neuronal splicing factors RBFOX1 and RBFOX2 in P19 cells with or without SRSF1 overexpression. The data show little influence of overexpressed SRSF1 on the expression of these splicing regulators during RA-induced P19 differentiation. (D) Immunofluorescence analysis of parental and SRSF1-overexpressing P19 cells induced by RA for 7 days. Anti-β-III tubulin antibody was used to demonstrate neural differentiation.

FIG. 7.

Model for collaboration between general and tissue-specific splicing regulators in regulated splicing. Strong SR protein interactions on the flanking constitutive exon 17 are responsible for skipping of the internal alternative exon 16. When the strong exon 17 is weakened, either by ESE mutations or by depletion of a trans-acting SR protein, the internal alternative exon 16 is selected, because of its proximity to the upstream constitutive exon 13. Inclusion of exon 16 is further enhanced by two intronic RBFOX binding events, which promote the upstream exon and inhibits the downstream exon, respectively. Downregulation of SR proteins in combination with other splicing regulators, such as RBFOX, may constitute a general strategy to achieve synergistic regulation of tissue-specific alternative splicing.