Global analysis of mRNA splicing

Abstract

Alternative mRNA splicing is a rich source of transcript diversity in eukaryotic cells with broad roles in development and disease. Systems-wide experimental methods have started to define how global splicing regulation shapes complex biological properties and pathways. Here, we review these approaches, describe recent insights they have yielded, and discuss avenues of future investigation.

FIGURE 1.

A hierarchy of splicing regulation. (A) Splicing patterns are a composite output of transcript-encoded cis elements and combinatorial binding of RNABPs to those elements. Shown are various upstream regulatory mechanisms that control the balance of nuclear RNABPs that regulate splicing decisions. These mechanisms are in turn responsive to signaling pathways that relay context-specific instructions. (B) The cellular organization of some regulatory mechanisms is shown. Signaling pathways affect levels, localization, and binding affinities of RNABPs via post-transcriptional mechanisms and post-translational modifications, including phosphorylation (P). Splicing patterns are determined by combinatorial binding of RNABPs to transcript-encoded cis elements (gray boxes).

FIGURE 2.

Microarray analysis of alternative splicing. Probe topographies for different array designs are schematized for a hypothetical transcript with one alternative (dark gray) and three constitutive (light gray) exons. Black probes are located wholly within exons, light gray and dark gray probes span exclusive exon junctions in isoforms a and b, respectively.

FIGURE 3.

Genome-wide profiling methods. (A) mRNA splicing begins during transcription in the nucleus, and finishes prior to nuclear export. RNABPs dynamically associate with transcripts throughout nuclear and cytoplasmic processing and transport steps. (B) Three methods to probe splicing regulation are shown, including ChIP and RIP analysis of a RNABP (black) and genome-wide transcript profiling. ChIP protocols cross-link (X) co-transcriptional processing factors to DNA, which is then profiled on microarrays. RIP involves purification of native or cross-linked RNPs and profiling associated transcripts. These methods can identify coregulated splicing networks. Expression profiling with splicing-sensitive microarrays generates a genome-wide survey of alternative splicing by measuring total, steady-state RNA.