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,...

The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3 splice sites (ss) of exon 2/2. Here, we demonstrate that exon 1 and 2/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3 ss and exon 1B to the proximal 3 ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter-driven splicing patterns but differed from 1A-promoter-driven splicing patterns, suggesting that promoter identity affects exon 2/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2/2 junction CAGAGAA, a sequence that overlaps the distal U2AF 35 -binding 3 ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3 ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3 ss in a transcript-dependent manner. Taken

The low prevalence of baby bottle images on commonly purchased baby gift and baby shower items is encouraging. However, the absence of breastfeeding images and the relatively high prevalence of baby dolls marketed with a baby bottle demonstrate that breastfeeding is not portrayed as the physiologic norm on these products. Product designers should explore ways to promote breastfeeding, consumers should make informed choices in product selection, and advocacy groups should promote guidelines for these products.