A Complex Network of Factors with Overlapping Affinities Repress Splicing through Intronic Elements

To better understand splicing regulation, we used a cell-based screen to identify ten diverse motifs that inhibit splicing from intron. Each motif was validated in another human cell type and gene context, and their presence correlated with in vivo splicing changes. All motifs exhibited exonic splicing enhancer or silencer activity, and grouping these motifs based on their distributions yielded clusters with distinct patterns of context-dependent activity. Candidate regulatory factors associated with each motif were identified, recovering 24 known and novel splicing regulators. Specific domains in selected factors were sufficient to confer ISS activity. Many factors bound multiple distinct motifs with similar affinity, and all motifs were recognized by multiple factors, revealing a complex, overlapping network of protein:RNA interactions. This arrangement enables individual cis-element to function differently in distinct cellular contexts depending on the spectrum of regulatory factors present.