Novel Splicing Factor RBM25 Modulates Bcl-x Pre-mRNA 5′ Splice Site Selection

Abstract

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...

ABA signalling is fine-tuned by antagonistic HAB1 variants

“…The sequence shows strong similarity to HsRBM25 (Supplementary Fig. 8b,c), a pre-mRNA splicing factor 31 . There is only a single copy of RBM25 in the Arabidopsis genome, and its homologues are ubiquitously expressed in eukaryotes (Supplementary Fig.…”

“…RBM proteins are important regulators in pre-mRNA splicing, and many RBM proteins are involved in the regulation of development and stress response 28 . In humans, HsRBM25 is a novel splicing factor that regulates pre-mRNA AS of an apoptotic factor Bcl-x producing the two isoforms, which have opposing roles during apoptosis 31 . We therefore hypothesized that the HsRBM25 homologue in Arabidopsis is involved in HAB1 AS.…”

“…RBM25 has been established as a novel splicing factor in humans. HsRBM25 regulates the AS of the apoptosis factor Bcl-x to produce the proapoptotic Bcl-xS and anti-apoptotic Bcl-xL isoforms, which have opposite roles in the regulation of apoptosis 31 . On the basis of this work, we suggest that Arabidopsis RBM25 regulates HAB1 AS ( Fig.…”

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins

“…Subsequently, investigations carried out by the Oesterreich group have related to repressive effects of SAFB on oestrogen receptor signalling (reviewed in Oesterreich [75] and Garee and Oesterreich [76]). As with many other RBPs [11,[77][78][79][80], overexpression of SAFB proteins produces generalised inhibition of RNA synthesis and eventual apoptosis [11,13]. This characteristic complicates interpretation of effects on gene expression associated with artificially raised levels of SAFB, though in some cases there is independent evidence that SAFB proteins are associated with gene silencing.…”

“…Thus, for example, overexpression of Sam68 [11,77], TIA-1 [78] and the RBM proteins [79,80] induces apoptosis, and the SAFB proteins do not appear to be an exception. Townson et al [103] showed that overexpression of SAFB1 inhibited cell growth and markedly decreased colony formation, whereas we found that overexpression of SLTM [13] and SAFB1 (unpublished observation, Uney, et al) causes apoptosis.…”