Defining the regulatory network of the tissue-specific splicing factors Fox-1 and Fox-2

Chaolin Zhang; Zuo Zhang; John Castle; Shuying Sun; Jason Johnson; Adrian R. Krainer; Michael Q. Zhang

doi:10.1101/gad.1703108

Defining the regulatory network of the tissue-specific splicing factors Fox-1 and Fox-2

¹ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA;
² Department of Biomedical Engineering, The State University of New York at Stony Brook, Stony Brook, New York 11794, USA;
³ Rosetta Inpharmatics LLC, a wholly owned subsidiary of Merck and Co., Seattle, Washington 98109, USA;
⁴ Department of Molecular and Cellular Biology, The State University of New York at Stony Brook, Stony Brook, New York 11794, USA

↵5 These authors contributed equally to this work.

Abstract

The precise regulation of many alternative splicing (AS) events by specific splicing factors is essential to determine tissue types and developmental stages. However, the molecular basis of tissue-specific AS regulation and the properties of splicing regulatory networks (SRNs) are poorly understood. Here we comprehensively predict the targets of the brain- and muscle-specific splicing factor Fox-1 (A2BP1) and its paralog Fox-2 (RBM9) and systematically define the corresponding SRNs genome-wide. Fox-1/2 are conserved from worm to human, and specifically recognize the RNA element UGCAUG. We integrate Fox-1/2-binding specificity with phylogenetic conservation, splicing microarray data, and additional computational and experimental characterization. We predict thousands of Fox-1/2 targets with conserved binding sites, at a false discovery rate (FDR) of ∼24%, including many validated experimentally, suggesting a surprisingly extensive SRN. The preferred position of the binding sites differs according to AS pattern, and determines either activation or repression of exon recognition by Fox-1/2. Many predicted targets are important for neuromuscular functions, and have been implicated in several genetic diseases. We also identified instances of binding site creation or loss in different vertebrate lineages and human populations, which likely reflect fine-tuning of gene expression regulation during evolution.

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