RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

Abstract

The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5= splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5= splice site, but not the engineered strong 5= splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5= splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5= splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5= splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.A lternative splicing is a eukaryotic regulatory mechanism that allows for the generation of numerous protein isoforms with often diverse biological functions from a single gene (4,26,41). It begins with the spliceosome, which is assembled stepwise by the addition of discrete small nuclear ribonucleoprotein particles (snRNPs) and numerous accessory non-snRNP splicing factors (23, 33). The excision of introns followed by the joining of exons depends on the recognition and usage of 5= and 3= splice sites (5= ss and 3= ss, respectively) by the splicing machinery (19, 34). The initial splicing step is comprised of 5= ss recognition by U1 snRNP and binding of U2 auxiliary factor (U2AF) to the 3= ss. These factors and additional proteins form the E or commitment complex, which bridges the intron and brings the splice sites close together. U2AF then recruits U2 snRNP to form the A complex. Subsequent binding of the U4-U6-U5 tri-snRNP and many other factors result in a fully assembled spliceosome that supports a series of rearrangements via RNA-RNA and RNA-protein interactions and activates the catalytic steps of cleavage, exon joining, and intron release (4, 26).The splice site signals that define the 5= ss and 3= ss of an alternatively spliced exon are often weak. How and when they are used is believed to be modulated by a complex interplay of positive (splicing enhancers) and negative (splicing silencers) cis elements and trans-acting factors (4, 26). These form the basis for alternative splicing. Target prediction for specific splicing factors is difficult, largely due to the small size and degeneracy of splicing factorbinding motifs. An exception to this degeneracy is the hexanucleotide UGCAUG, which has been shown to be an important element for the splicing of several exons (3,5,14,16,20,24,...

RBFOX2 Is an Important Regulator of Mesenchymal Tissue-Specific Splicing in both Normal and Cancer Tissues

“…However, RBFOX2-inhibited exons were either up-or downregulated in the mesenchyme (Fig. 5a), suggesting that RBFOX2 may be sufficient to promote exon inclusion but that exon skipping in mesenchyme may require an interplay between RBFOX2 and other factors, as suggested recently (49,50) and consistent with recent reports indicating that many RBFOX protein-regulated exons are also targets of other splicing factors, including Nova, PTBP, and ESRP proteins (26,(51)(52)(53).…”

The BRCA1-Δ11q Alternative Splice Isoform Bypasses Germline Mutations and Promotes Therapeutic Resistance to PARP Inhibition and Cisplatin

“…We first identified two binding elements for the FOX2 splicing site selection factor at position +18 and +63 downstream from the cryptic splice site location (Supplementary Fig. S6) (44, 45). Depletion of FOX2 using 2 individual siRNAs did not impact +11 expression levels, but reduced Δ11q expression 1.7- ( P = 0.0043) and 1.6-fold ( P = 0.0179) in MDA-MB-231, 1.5- ( P = 0.048) and 1.6-fold ( P = 0.0301) in UWB1.289, 2.2- ( P = 0.0056) and 3.1-fold ( P = 0.0017) in SUM149PT cells, compared to scrambled siRNA treated cells, respectively (Fig.…”

Rbfox Proteins Regulate Splicing as Part of a Large Multiprotein Complex LASR

“…Instead of Rbfox the microprocessor contains Drosha and DGCR8 that carry out miRNA processing. Other described Rbfox interactions include with U1C, hnRNP K, Sam68, RALY, PSF, TFG, and Ataxin2, as well as the aforementioned hnRNP H contact (Huang et al, 2012; Kim et al, 2011; Mauger et al, 2008; Shibata et al, 2000; Sun et al, 2012). Recombinant Rbfox added to an in vitro splicing extract inhibited assembly of a pre-spliceosomal E-complex on an Rbfox-repressed exon (Fukumura et al, 2007; Zhou and Lou, 2008).…”