Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

Abstract

Over 95% of human multi-exon genes undergo alternative splicing, a process important in normal development and often dysregulated in disease. We sought to analyze the global splicing regulatory network of CELF2 in human T cells, a well-studied splicing regulator critical to T cell development and function. By integrating high-throughput sequencing data for binding and splicing quantification with sequence features and probabilistic splicing code models, we find evidence of splicing antagonism between CELF2 and the RBFOX family of splicing factors.We validate this functional antagonism through knockdown and overexpression experiments in human cells and find CELF2 represses RBFOX2 mRNA and protein levels. Because both families of proteins have been implicated in the development and maintenance of neuronal, muscle, and heart tissues, we analyzed publicly available data in these systems. Our analysis suggests global, antagonistic co-regulation of splicing by the CELF and RBFOX proteins in mouse muscle and heart in several physiologically relevant targets including proteins involved in calcium signaling and members of the MEF2 family of transcription factors. Importantly, a number of these co-regulated events are aberrantly spliced in mouse models and human patients with diseases that affect these tissues including heart failure, diabetes, or myotonic dystrophy. Finally, analysis of exons regulated by ancient CELF family homologs in chicken, and Drosophila suggests this antagonism is conserved through evolution.

Rbfox1 is required for myofibril development and maintaining fiber-type specific isoform expression inDrosophilamuscles

“…Rbfox binding motifs are found to be co-enriched with MBNL and CELF motifs around the same groups of exons in human, mouse and chicken (Bland et al, 2010;Kalsotra et al, 2008;Merkin et al, 2012). Rbfox1 and MBNL co-regulate a significant number of alternative events altered in DM1 skeletal muscle (Klinck et al, 2014), while CELF2 and Rbfox2 co-regulate and co-bind introns flanking exons regulated in cardiac development or with altered expression in hearts of a Type I diabetes mouse model (Gazzara et al, 2017). CELF2 moreover represses Rbfox2 expression in heart, and overexpression of CELF1/2 or depletion of Rbfox2 leads to the same changes in splicing direction and magnitude (Gazzara et al, 2017).…”

“…Many developmentally-regulated, alternatively spliced exons in vertebrate muscle have binding sites for both FOX and CELF family RNA-binding proteins, and in heart notably appear to be antagonistically co-regulated by CELF2 and RBFOX2 (Bland et al, 2010;Gazzara et al, 2017). Thus, we next checked if Rbfox1 and Bru1 co-regulate alternative splicing in Drosophila muscle.…”

“…Rbfox1 and MBNL co-regulate a significant number of alternative events altered in DM1 skeletal muscle (Klinck et al, 2014), while CELF2 and Rbfox2 co-regulate and co-bind introns flanking exons regulated in cardiac development or with altered expression in hearts of a Type I diabetes mouse model (Gazzara et al, 2017). CELF2 moreover represses Rbfox2 expression in heart, and overexpression of CELF1/2 or depletion of Rbfox2 leads to the same changes in splicing direction and magnitude (Gazzara et al, 2017). We see evidence for similar regulatory interactions between Rbfox1 and the CELF1/2 homolog Bru1 in our data from Drosophila.…”

Integrative Deep Models for Alternative Splicing

“…Additionally, a number of Rbfox binding motifs (GCAUG) and CLIP peaks are located just downstream of these exons (Fig 4c, right), where these proteins enhance inclusion [13]. These observations motivated additional study in human T cells where we found Celf2 is a potent repressor of Rbfox2 [6], suggesting that a similar indirect mechanism may be at play in murine muscle and heart where Celf overexpression represses Rbfox proteins to drive splicing changes in these and other targets.…”

Nucleocytoplasmic Transport of RNA-Binding Proteins Regulates Neural Stem Cell Fates 

“…The combinatorial functions of CELF2 and its interactions with other RBPs (e.g. RBFOX) might allow an elaborate regulation of NPC fates through the precise control of mRNAs at different stages of gene expression (Gazzara et al, 2017;Zhang et al, 2016).…”