Splicing segregation: the minor spliceosome acts outside the nucleus and controls cell proliferation.

The functional relevance and the evolution of two parallel mRNA splicing systems in eukaryotes--a major and minor spliceosome that differ in abundance and splicing rate--are poorly understood. We report here that partially spliced pre-mRNAs containing minor-class introns undergo nuclear export and that minor-class snRNAs are predominantly cytoplasmic in vertebrates. Cytoplasmic interference with the minor spliceosome further indicated its functional segregation from the nucleus. In keeping with this, minor splicing was only weakly affected during mitosis. By selectively interfering with snRNA function in zebrafish development and in mammalian cells, we revealed a conserved role for minor splicing in cell-cycle progression. We argue that the segregation of the splicing systems allows for processing of partially unspliced cytoplasmic transcripts, emerging as a result of different splicing rates. The segregation offers a mechanism accounting for spliceosome evolution in a single lineage and provides a means for nucleus-independent control of gene expression.

Targeted 'knockdown' of spliceosome function in mammalian cells.

The existence of two sophisticated parallel splicing machineries in multicellular organisms has raised intriguing questions--ranging from their impact on proteome expansion to the evolution of splicing and of metazoan genomes. Exploring roles for the distinct splicing systems in vivo has, however, been restricted by the lack of techniques to selectively inhibit their function in cells. In this study, we show that morpholino oligomers complementary to the branch-site recognition elements of U2 or U12 small nuclear RNA specifically suppress the function of the two splicing systems in mammalian cells. The data provide the first evidence for a role of distinct spliceosomes in pre-mRNA splicing from endogenous mammalian genes and establish a tool to define roles for the different splicing machineries in vivo.

Signal-dependent regulation of splicing via phosphorylation of Sam68.

Evolution of human organismal complexity from a relatively small number of genes--only approximately twice that of worm or fly--is explained mainly by mechanisms generating multiple proteins from a single gene, the most prevalent of which is alternative pre-messenger-RNA splicing. Appropriate spatial and temporal generation of splice variants demands that alternative splicing be subject to extensive regulation, similar to transcriptional control. Activation by extracellular cues of several cellular signalling pathways can indeed regulate alternative splicing. Here we address the link between signal transduction and splice regulation. We show that the nuclear RNA-binding protein Sam68 is a new extracellular signal-regulated kinase (ERK) target. It binds exonic splice-regulatory elements of an alternatively spliced exon that is physiologically regulated by the Ras signalling pathway, namely exon v5 of CD44. Forced expression of Sam68 enhanced ERK-mediated inclusion of the v5-exon sequence in mRNA. This enhancement was impaired by mutation of ERK-phosphorylation sites in Sam68, whereas ERK phosphorylation of Sam68 stimulated splicing of the v5 exon in vitro. Finally, Ras-pathway-induced alternative splicing of the endogenous CD44-v5 exon was abolished by suppression of Sam68 expression. Our data define Sam68 as a prototype regulator of alternative splicing whose function depends on protein modification in response to extracellular cues.