No abstract

Bcl-x is a member of the Bcl-2 family of proteins that are key regulators of apoptosis. The Bcl-x pre-mRNA is alternatively spliced to yield Bcl-x S and Bcl-x L , two isoforms that have been associated, respectively, with the promotion and the prevention of apoptosis. We have investigated some of the elements and factors involved in the production of these two splice variants. Deletion mutagenesis using a human Bcl-x minigene identifies two regions in exon 2 that modulate Bcl-

hnRNP A/B proteins modulate the alternative splicing of several mammalian and viral pre-mRNAs, and are typically viewed as proteins that enforce the activity of splicing silencers. Here we show that intronic hnRNP A/B–binding sites (ABS) can stimulate the in vitro splicing of pre-mRNAs containing artificially enlarged introns. Stimulation of in vitro splicing could also be obtained by providing intronic ABS in trans through the use of antisense oligonucleotides containing a non-hybridizing ABS-carrying tail. ABS-tailed oligonucleotides also improved the in vivo inclusion of an alternative exon flanked by an enlarged intron. Notably, binding sites for hnRNP F/H proteins (FBS) replicate the activity of ABS by improving the splicing of an enlarged intron and by modulating 5′ splice-site selection. One hypothesis formulated to explain these effects is that bound hnRNP proteins self-interact to bring in closer proximity the external pair of splice sites. Consistent with this model, positioning FBS or ABS at both ends of an intron was required to stimulate splicing of some pre-mRNAs. In addition, a computational analysis of the configuration of putative FBS and ABS located at the ends of introns supports the view that these motifs have evolved to support cooperative interactions. Our results document a positive role for the hnRNP A/B and hnRNP F/H proteins in generic splicing, and suggest that these proteins may modulate the conformation of mammalian pre-mRNAs.

The heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in the regulation of mRNA metabolism by specifically recognizing G-tract RNA sequences. We have determined the solution structures of the three quasi RNA recognition motifs (qRRMs) of hnRNP F in complex with G-tract RNA. These structures show that qRRMs bind RNA in a very unusual manner, the G-tract being "encaged", making the qRRM a novel RNA binding domain. We defined a consensus signature sequence for qRRMs and identified other human qRRM-containing proteins, which also specifically recognize G-tract RNAs. Our structures explain how qRRMs can sequester G-tracts maintaining them in a single-stranded conformation. We also show that isolated qRRMs of hnRNP F are sufficient to regulate the alternative splicing of the Bcl-x premRNA strongly suggesting that hnRNP F would act by remodeling RNA secondary and tertiary structures.3

β-site APP cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step in the proteolysis of the amyloid β-protein precursor (APP) to the amyloid β-protein (Aβ), a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, the regulation of which is not well understood. We identified a G-rich sequence within exon 3 of BACE1 involved in controlling splice site selection. Mutation of the G-rich sequence decreased use of the normal 5′ splice site of exon 3, which leads to full-length and proteolytically active BACE1, and increased use of an alternative splice site, which leads to a shorter, essentially inactive isoform. Nuclease protection assays, nuclear magnetic resonance, and circular dichroism spectroscopy revealed that this sequence folds into a G-quadruplex structure. Several proteins were identified as capable of binding to the G-rich sequence, and one of these, heterogeneous nuclear ribonucleoprotein H (hnRNP H), was found to regulate BACE1 exon 3 alternative splicing and in a manner dependent on the G-rich sequence. Knockdown of hnRNP H led to a decrease in the full-length BACE1 mRNA isoform as well as a decrease in Aβ production from APP, suggesting new possibilities for therapeutic approaches to AD.