Large-scale analysis of gene expression data has revealed that most human genes have the capacity to encode multiple proteins through the process of alternative splicing (Pan et al., 2008
; Wang et al., 2008
). Importantly, variant proteins expressed from a single gene via alternative splicing often act in competition or opposition to one another, such that even small changes in the ratio of protein isoforms expressed from a given gene can have a dramatic physiologic effect (Matlin et al., 2005
). Therefore, the proteins and mechanisms that control alternative splicing play a critical role in determining protein expression and cellular function.
The catalysis of pre-mRNA splicing is mediated by the “spliceosome” — a macromolecular machine comprised of five small nuclear RNAs (U1, U2, U4, U5, U6 snRNA) and associated proteins that interact with sequences at the exon/intron boundaries (“splice sites”) to direct the excision of introns and ligation of exons (Wahl et al., 2009
). The catalytic spliceosome (C complex) is not a pre-formed enzyme, but rather assembles on the pre-mRNA in a stepwise pathway that involves several distinct intermediates (E-A-B complexes; Wahl et al. 2009
). In higher eukaryotes, the splice site sequences are highly degenerate and alone do not typically contain sufficient information to accurately determine the sites of cleavage and ligation (Black, 1995
; Matlin et al., 2005
). It is now widely established that the binding of an exon by the spliceosome is typically controlled by various proteins bound to auxiliary sequences located within exons or flanking introns (Matlin et al., 2005
). Interestingly, in many cases the same splicing regulatory protein can enhance inclusion of some exons while promoting skipping of others, although the mechanisms by which such dual effects are conferred remain poorly understood in most cases.
An emerging theme in alternative splicing is that of networks of co-regulated events, in which a single protein coordinates the inclusion or exclusion of exons in multiple genes. For example, coordinate regulation has been demonstrated for genes involved in controlling synaptic plasticity via the neural-specific protein Nova (Ule et al., 2006
). Similarly, the neural and muscle-specific proteins Fox-1/2 regulate the splicing of multiple genes involved in neuromuscular function (Zhang et al., 2008
). These studies, among others, have introduced the notion of regulatory “maps” that predict the effect of a protein based on location of binding. Critically, however, two inherent assumptions of these maps have not yet been tested. First, does a given protein always functions by the same mechanism when bound to a particular location relative to an exon and, second, is location the sole determinant of mechanism, or can other variables influence how a particular protein functions?
A well studied example of regulated alternative splicing is the CD45 gene, which has three variable exons (4, 5 and 6) that are coordinately skipped upon antigen-induced activation of T cells (Hermiston et al., 2002
). Skipping of the CD45 variable exons is regulated, at least in part, by binding of hnRNP L to an activation-responsive sequence (ARS) that is located within each variable exon (Rothrock et al., 2003
; Rothrock et al., 2005
; Tong et al., 2005
). For exons 4 and 6 the ARS motif is embedded within a 60 nt exonic splicing silencer (ESS1) element that is both necessary and sufficient for regulation (Rothrock et al., 2003
; Tong et al., 2005
). In contrast, the ARS motif in exon 5 is split across two regions (S1 and S2
) that are separated by an exonic splicing enhancer sequence (ESE) (Tong et al., 2005
; ). Therefore comparison of the regulation of CD45 exons 4 and 5 provides a powerful system for determining whether the broader sequence context of an exon can influence the mechanisms by which a particular regulatory element and/or associated proteins functions.
Differential arrangement of the ARS regulatory element in the three variable exons (4, 5, and 6) of the CD45 gene
In this study we show that hnRNP L can repress or activate an exon by distinct mechanisms due, at least in part, to differences in splice site strength. Binding and functional studies demonstrate that hnRNP L bound to the silencers in exon 5 directly competes with SF2/ASF bound to an ESE, inhibiting the ability of the ESE-complex to recruit the U2snRNP to the weak upstream 3’ss. This mechanism is markedly distinct from the previously reported mechanism of direct repression of exon 4 by hnRNP L (House and Lynch, 2006
). Because the splice sites flanking exon 5 are weak compared to those of exon 4, we directly examined the effect of hnRNP L binding to exons with varying splice site strengths. Remarkably, in multiple distinct exon contexts we find that hnRNP L represses strong splice sites but enhances weak splice sites. These data provide direct evidence that a given protein can function through different mechanisms in a manner independent of location but constrained by the local sequence context. We propose a unifying model for hnRNP L function in which stabilization of U1 and U2 snRNP binding promotes assembly on weak splice sites or across an intron, but traps these snRNPs in a inactive complex when hnRNP L is bound to an exon flanked by strong splice sites.