Global Promotion of Alternative Internal Exon Usage by mRNA 3' End Formation Factors

Abstract

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.

Figure 1. A Large-Scale shRNA Screen Reveals CPSF as an RBFOX2 Cofactor

(A) Schematic of the screen. (B) FACS analysis showing the percentage of GFP- cells in GFP/Flp-In-293 cells expressing a non-silencing (NS), RBFOX2 or CPSF2 shRNA. (C) RT-PCR analysis of GFP splicing in parental (FACS sorted) GFP/Flp-In-293 cells, and cells expressing a NS, RBFOX2 or CPSF2 shRNA. The positions of mRNAs in which exon 2 is included (E2-I) or excluded (E2-E) is shown. The percentage of the signal corresponding to exon inclusion is shown. (D and E) RT-PCR analysis of GFP splicing in GFP/Flp-In-293 cells (D) and of endogenous MBNL1 in Flip-In-293 cells (E) expressing a control NS shRNA, or an shRNA targeting an mRNA 3’-end formation factor. (F) CLIP analysis monitoring binding of RBFOX2, CPSF1–4, SYMPK and other mRNA 3’-end formation factors to MBNL1 exon 8. Data are represented as mean ± SD. (G) CLIP analysis monitoring binding of RBFOX2, CPSF1–4 and SYMPK to MBNL1 exon 8 in cells expressing a NS, RBFOX2, CPSF1–4 or SYMPK shRNA. Data are represented as mean ± SD. (H) RNA pull-down assay. A biotinylated RNA containing MBNL1 regulated exon 8 (E8-WT), an exon 8 derivative containing a mutation in the RBFOX2-binding site (E8-mut) or, as a control, constitutively-spliced exon 6 [E6 (ctrl)] as well as 100 bp of upstream and downstream intron sequences was incubated in nuclear extract and analyzed for bound proteins by immunoblotting. (I) Co-immunoprecipitation analysis. Cell extracts were immunoprecipitated in the presence of RNase with an RBFOX2, CPSF1–4 or SYMPK antibody, or as a control IgG, and immunoblotted for each protein. See also Figure S1.

Figure 2. Genome-Wide Analysis of RBFOX2- and CPSF2-Mediated Changes in Alternative Splicing of Internal Exons

(A) Venn diagrams showing the overlap between total (left), exon exclusion (middle) and exon inclusion (right) RBFOX2-regulated and CPSF2-regulated internal splicing events. (B) Distribution of RBFOX2 and CPSF2 iCLIP tags. (C) Top binding motifs, based on P values, from RBFOX2 and CPSF2 iCLIP analysis. (D) RNA pull-down assay. A biotinylated RNA substrate containing β-globin exon 2 harboring (E2+CPSF2) or lacking (E2-WT; control) two AGGUAG motifs was incubated with in vitro translated proteins and analyzed for bound proteins by immunoblotting. See also Figures S2 and S7.

Figure 3. Validation of RNA-Seq Results for RBFOX2-Regulated, CPSF/SYMPK-Regulated Exons

(A and B) RT-PCR analysis monitoring inclusion (A) or exclusion (B) of representative exons in cells expressing a NS, RBFOX2, CPSF1–4 or SYMPK shRNA. (C and D) CLIP analysis monitoring binding of RBFOX2, CPSF1–4 and SYMPK to the regulated exons of the indicated pre-mRNAs. Data are represented as mean ± SD. (E) CLIP analysis monitoring binding of RBFOX2, CPSF1–4 and SYMPK to MEF2A exon 8 in cells expressing a NS, RBFOX2, CPSF1–4 or SYMPK shRNA. Data are represented as mean ± SD. See also Figure S3.

Figure 4. Validation of RNA-Seq Results for CPSF/SYMPK-Regulated, RBFOX2-Independent Exons

(A and B) RT-PCR analysis monitoring inclusion (A) or exclusion (B) of representative exons in cells expressing a NS, RBFOX2, CPSF1–4 or SYMPK shRNA. (C and D) CLIP analysis monitoring binding of RBFOX2, CPSF1–4 and SYMPK to the regulated exons of the indicated pre-mRNAs. Data are represented as mean ± SD. See also Figure S4.

Figure 5. RBFOX2 and CPSF/SYMPK Promote Exon Exclusion and Inclusion by Regulating Binding of U2AF and U1 snRNP

(A and B) CLIP analysis monitoring binding of U2AF65 and U1 70K to regulated and constitutive exons in MEF2A (A) and MBNL1 (B) in cells expressing a NS, RBFOX2 or CPSF2 shRNA. Data are represented as mean ± SD. (C) Co-immunoprecipitation analysis. Cell extracts were immunoprecipitated in the presence of RNase with an RBFOX2 or CPSF2 antibody, or as a control IgG, and immunoblotted for RBFOX2, CPSF2, U2AF65 or U1 70K. (D) RNA pull-down assays monitoring binding of RBFOX2, CPSF2, U2AF65 and U1 70K to a biotinylated RNA substrate containing MEF2A exon 8 (inclusion) or MBNL1 exon 8 (exclusion) in extracts prepared from Flp-In-293 cells expressing an NS, RBFOX2 or CPSF2 shRNA. (E and F) RNA pull-down assays monitoring binding of RBFOX2, CPSF2, U2AF65 and U1 70K to a biotinylated RNA substrate containing a wild-type MEF2A exon 8 (E) or MBNL1 exon 8 (F) or mutant derivative in which the RBFOX2-binding site, CPSF2-binding site, Py tract/3’ splice site or 5’ splice site was mutated. See also Figure S5.

Figure 6. CPSF/SYMPK is also a Cofactor for the Splicing Regulators NOVA2 and HNRNPA1

(A) (Left) RT-PCR analysis monitoring exon inclusion (TRIO) or exclusion (TNRC6A) in cells expressing a NS, NOVA2, CPSF1–4 or SYMPK shRNA. (Right) iCLIP tracks. (B) CLIP analysis monitoring binding of NOVA2, CPSF1–4 and SYMPK to the regulated exons of the indicated pre-mRNAs. Data are represented as mean ± SD. (C) CLIP analysis monitoring binding of NOVA2, CPSF1–4 and SYMPK to TRIO exon 36 in cells expressing a NS, NOVA2, CPSF1–4 or SYMPK shRNA. Data are represented as mean ± SD. (D) (Left) RT-PCR analysis monitoring exon inclusion (MAPK45) or exclusion (UFL1) in cells expressing a NS, HNRNPA1, CPSF1–4 or SYMPK shRNA. (Right) iCLIP tracks. (E) CLIP analysis monitoring binding of HNRNPA1, CPSF1–4 and SYMPK to the regulated exons of the indicated pre-mRNAs. Data are represented as mean ± SD. (F) CLIP analysis monitoring binding of HNRNPA1, CPSF1–4 and SYMPK to UFL1 exon 12 in cells expressing a NS, HNRNPA1, CPSF1–4 or SYMPK shRNA. Data are represented as mean ± SD. See also Figure S6.

Figure 7. NOVA2 and HNRNPA1 Promote Exon Inclusion and Exclusion by a Mechanism Similar to that of RBFOX2

(A) RNA pull-down assay. A biotinylated RNA containing TRIO regulated exon 36 (left) or UFL1 regulated exon 12 (right), derivatives containing a mutation in the NOVA2- or HNRNPA1-binding site or, as a control, a constitutively-spliced exon (exon 34 of TRIO or exon 6 of UFL1) as well as 100 bp of upstream and downstream intron sequences was incubated in nuclear extract and analyzed for bound proteins by immunoblotting. (B and C) Co-immunopreciptation analysis. Cell extracts were immunoprecipitated in the presence of RNase with a NOVA2 (B) or HNRNPA1 (C) antibody, or CPSF1–4 or SYMPK antibody, or an IgG control, and immunoblotted for each protein. (D) CLIP analysis monitoring binding of U2AF65 and U1 70K to regulated and constitutive exons in TRIO (top) and TNRC6A (bottom) in cells expressing a NS, NOVA2 or CPSF2 shRNA. Data are represented as mean ± SD. (E) CLIP analysis monitoring binding of U2AF65 and U1 70K to regulated and constitutive exons in MAP4K5 (top) and UFL1 (bottom) in cells expressing a NS, HNRNPA1 or CPSF2 shRNA. Data are represented as mean ± SD. (F) Model for CPSF/SYMPK-mediated regulation of alternative splicing of internal exons. See also Figure S6.