Rbfox2 controls autoregulation in RNA-binding protein networks

Abstract

The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsensemediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to highthroughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These ''silent'' events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMDinducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.

Alternative Splicing in the Mammalian Nervous System: Recent Insights into Mechanisms and Functional Roles

“…A significant fraction of Rbfox-regulated AS events that exhibit dynamic changes in inclusion levels during brain development were also identified (Weyn-Vanhentenryck et al, 2014). These studies complement and extend previous investigations of the networks of exons regulated by Rbfox proteins (Jangi et al, 2014;Yeo et al, 2009;Zhang et al, 2008).…”

“…This mechanism for suppressing Ptpb1 function contributes to the inclusion of the neural-specific Ptbp2 exon, thus facilitating Ptbp2 expression at a later stage during neural development (Boutz et al, 2007;Spellman et al, 2007). Ptbp2 is also subject to autoregulation and cross-regulation by Rbfox2 via AS coupled to NMD (Jangi et al, 2014).…”

The PUF binding landscape in metazoan germ cells

“…ife-1 encodes a germline-specific eIF4E translation factor and promotes GSC differentiation (Henderson et al 2009). The 3 ′ UTR peaks in fbf-1 and fbf-2 mRNAs are consistent with autoregulation, as seen for other RNA binding proteins including other PUF proteins (Lamont et al 2004;Dredge et al 2005;Buratti and Baralle 2011;Anko et al 2012;Weidmann and Goldstrohm 2012;Jangi et al 2014).…”

Induced transcription and stability of CELF2 mRNA drives widespread alternative splicing during T-cell signaling

“…Crossregulation of splicing factors has been described in a number of studies (14,37,53,54), suggesting that splicing factors represent a tightly knit network of positive and negative regulation that finely shapes overall transcriptome processing. The results we present here suggest that CELF2 may be a key hub in this regulatory network, and that CELF2's impact on global mRNA processing may include indirect as well as direct effects.…”