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.

High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 59 splice site in mammalian premRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 59 splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 59 splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 59 splice site usage in an equivalent manner. Second, increasing the distance between the internal 59 splice site and flanking A1 binding sites does not compromise activation of the distal 59 splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 59 splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 59 splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 59 splice site and lead to distal 59 splice site activation.

Depending on the cell lines and cell types, dimethyl sulfoxide (Me 2 SO) can induce or block cell differentiation and apoptosis. Although Me 2 SO treatment alters many levels of gene expression, the molecular processes that are directly affected by Me 2 SO have not been clearly identified. Here, we report that Me 2 SO affects splice site selection on model pre-mRNAs incubated in a nuclear extract prepared from HeLa cells. A shift toward the proximal pair of splice sites was observed on pre-mRNAs carrying competing 5-splice sites or competing 3-splice sites. Because the activity of recombinant hnRNP A1 protein was similar when added to extracts containing or lacking Me 2 SO, the activity of endogenous A1 proteins is probably not affected by Me 2 SO. Notably, in a manner reminiscent of SR proteins, Me 2 SO activated splicing in a HeLa S100 extract. Moreover, the activity of recombinant SR proteins in splice site selection in vitro was improved by Me 2 SO. Polar solvents like DMF and formamide similarly modulated splice site selection in vitro but formamide did not activate a HeLa S100 extract. We propose that Me 2 SO improves ionic interactions between splicing factors that contain RS-domains. The direct impact of Me 2 SO on alternative splicing may explain, at least in part, the different and sometimes opposite effects of Me 2 SO on cell differentiation and apoptosis.Me 2 SO 1 is a polar solvent used to promote cell differentiation of tumor cell lines. For example, the treatment of mouse erythroleukemic and neuroblastoma cells with 2% Me 2 SO induces morphological changes and differentiation in red blood cells and neurons, respectively (e.g. see Refs. 1, 2). Me 2 SO also induces differentiation of the human U937 monoblast leukemia cell line into monocyte/macrophage (3) and stimulates the differentiation of a human ovarian adenocarcinoma cell line (4). Paradoxically, Me 2 SO prevents the terminal differentiation of myoblasts (5, 6), inhibits the differentiation of adipocytes (7), blocks the differentiation of antibody-producing plasma cells (8), and interferes with the differentiation of chick embryo chondrocytes (9). Whereas Me 2 SO has been used to induce apoptosis in some cell lines (10, 11), it inhibits cell density-dependent apoptosis of CHO cells (6). Thus, depending on the cell line, Me 2 SO can have completely different effects on differentiation and apoptosis.The cellular mechanisms that are affected by Me 2 SO remain unclear. Because Me 2 SO facilitates DNA uptake during transfection procedures (e.g. see Ref. 12), Me 2 SO has been proposed to affect the integrity of cell membranes. Because Me 2 SO alters protein kinase C activity and the expression of integrin complexes (6, 13), Me 2 SO may alter intracellular signaling processes, which in turn may have a broad impact on many aspects of gene expression. Me 2 SO treatment promotes changes in the abundance of certain mRNAs and in the ratio of spliced isoforms (14 -17). Among the genes reported to be affected in their alternative splicing is the NCAM pre-mR...