RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-x S 5 splice site (5 ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-x L . Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-x S 5 ss selection, leading to decreased Bcl-x S isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5 ss; however, it was not required when a strong consensus 5 ss was present. In support of a role for RBM25 in modulating the selection of a 5 ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-x S 5 ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.Alternative splicing is a regulatory mechanism that allows eukaryotes to generate numerous protein isoforms, often with diverse biological functions, from a single gene (2,26,49). Pre-mRNA splicing takes place within the spliceosome, which is assembled stepwise by the addition of small nuclear ribonucleoprotein particles (snRNP) and numerous accessory nonsnRNP splicing factors (23, 31). The excision of introns and the joining of exons depend on the recognition and usage of 5Ј splice sites (5Ј ss) and 3Ј ss by the splicing machinery (21, 33). The commitment complex forms when the 5Ј ss is recognized by U1 snRNA base pairing and stabilized by U1 snRNP while the 3Ј ss is recognized by the U2 auxiliary factor (U2AF) through U2 snRNA base pairing with the branch point. Subsequently, the U4/5/6 tri-snRNP is incorporated into the complex and the U1 snRNA base paired at the 5Ј ss is replaced by U6 snRNA. These processes 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 (2, 26).However, the splice site signals that define the 5Ј ss and 3Ј ss are often degenerate. How and when they are used are believed to be modulated by a combinational interplay of positive (splicing enhancers) and negative (splicing silencers) cis elements and trans-acting factors (2, 26), forming the basis of alternative splicing. The splicing regulatory (SR) proteins (18, 41) and the heterogeneous nuclear ribonucleoproteins (hnRNPs) (11,46) bind with specificity to pre-mRNA (20).The SR proteins generally bind to enhancer elements through the RNA-binding domain and activate splicing at...

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

Many protein factors that guide pre-mRNA modification pathways are composed of RNA recognition motif (RRM) domains. In an effort to identify splicing regulators in erythroid cells, we cloned a RRM-containing protein RBM25 and characterized its role in alternative splicing. RBM25 consists of a proline-rich region and a RRM domain at the amino-terminal end, an ER rich domain at the central region, and a PWI domain at the carboxyl terminal end. RBM25 partially co-localized with splicing factor SC35 in nuclear speckles. While both the RRM and PWI domains are diffusely distributed in the nucleoplasm, the ER domain is highly concentrated in nuclear speckles. We examined cellular mRNA targets of RBM25 in HeLa cells and demonstrated that it binds Bcl-x mRNA and affects its alternative splicing. Depletion of RBM25 by RNA interference caused accumulation of anti-apoptotic Bcl-x(L), whereas its up-regulation increased the levels of pro-apoptotic Bcl-x(s). The expression level of RBM25 also correlated with the degree of cell death, further suggesting that it functions in regulating apoptotic factor(s) expression. Apoptosis plays an important role in red cell development; earlier erythroid progenitors are more sensitive to apoptosis while mature erythroblasts are resistant to apoptosis. A significant decrease in RBM25 expression occurs during erythroid differentiation and correlates with resistance to apoptosis. Our results suggest that erythroblasts may acquire resistance to apoptosis during maturation through differential expression of crucial splicing regulators of the apoptotic machinery.