Understanding alternative splicing: towards a cellular code

Abstract

Alternative splicing allows individual genes to generate multiple mRNAs. Many of these mRNAs encode functionally distinct protein isoforms, thereby bridging the gap between genome and proteome. Alternative splicing mechanisms have traditionally been investigated using individual model systems, but these approaches are now being complemented by global analyses. The regulation of splicing commonly involves the modulation of early steps in spliceosome assembly. The cis elements comprise splicing enhancers and silencers that can be located in either the exons or the introns and that bind activator and repressor proteins. Sometimes, the presence or absence of a single regulator is sufficient to determine alternative splicing pathways. More commonly, combinations of more widespread factors are involved in the choice of splicing pathway. This gives rise to the concept of 'cellular codes', which are constituted by particular combinations of regulatory factors. Members of the SR protein family can activate splicing by binding to exon splicing enhancers (ESEs). Models for the action of enhancers include the recruitment of the U2 auxiliary factor (U2AF) to weak 3′ splice sites, the interaction with co-activators and direct contact with the RNA at the branch point. Repressors, which are frequently members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, function by making splice sites inaccessible or by promoting the formation of 'dead-end' splicing related complexes. Alternative splicing decisions frequently involve the dynamic antagonism between regulatory activators and repressors. Several global strategies for identifying splicing silencers and enhancers have been successfully deployed. These include RNA-binding SELEX studies, functional SELEX in vitro, cell-based selection assays and computational surveys for the differential enrichment of sequence motifs in expected locations for silencers and enhancers. Methods are being developed to detect complete sets of cellular RNAs with which individual splicing regulators interact. These include assays that detect RNAs to which the factor binds, or alternative splicing events that are affected by depletion of the regulator. A key technical development is the recent introduction of alternative splicing microarrays, which allow parallel analysis of large numbers of alternative splicing events and should facilitate the deciphering of cellular codes.