Regulation of alternative splicing: more than just the ABCs.

Alternative pre-mRNA splicing, the differential inclusion or exclusion of portions of a nascent transcript into the final protein-coding mRNA, is widely recognized to be a ubiquitous mechanism for controlling protein expression. Thus, understanding the molecular basis of alternative splicing is essential for deciphering post-transcriptional control of the genome. Pre-mRNA splicing in general is catalyzed by a large dynamic macromolecular machine known as the spliceosome. Notably, the recognition of the intron substrate by spliceosomal components and the assembly of these components to form a catalytic spliceosome occur through a network of highly combinatorial molecular interactions. Many, if not all, of these interactions are subject to regulation, forming the basis of alternative splicing. This minireview focuses on recent advances in our understanding of the diversity of mechanisms by which the spliceosome can be regulated so as to achieve precise control of alternative splicing under a range of cellular conditions.

An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition.

Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosome's dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4-U6-U5 tri-snRNP-containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway.

HnRNP L represses exon splicing via a regulated exonic splicing silencer.

Skipping of mammalian exons during pre-mRNA splicing is commonly mediated by the activity of exonic splicing silencers (ESSs). We have recently identified a regulated ESS within variable exon 4 of the CD45 gene, named ESS1, that is necessary and sufficient for partial exon repression in resting T cells and has additional silencing activity upon T-cell activation. In this study, we identify three heterogeneous nuclear ribonucleoproteins (hnRNPs) that bind specifically to ESS1. The binding of one of these proteins, hnRNP-L, is significantly decreased by mutations that disrupt both the basal and induced activities of ESS1. Recombinant hnRNP-L functions to repress exon inclusion in vitro in an ESS1-dependent manner. Moreover, depletion of hnRNP-L, either in vitro or in vivo, leads to increased exon inclusion. In contrast, the other ESS1-binding proteins, PTB and hnRNP E2, do not discriminate between wild-type and mutant ESS1 in binding studies, and do not specifically alter ESS1-dependent splicing in vitro. Together, these studies demonstrate that hnRNP-L is the primary protein through which CD45 exon 4 silencing is mediated by the regulatory sequence ESS1.