Activation-Dependent TRAF3 Exon 8 Alternative Splicing Is Controlled by CELF2 and hnRNP C Binding to an Upstream Intronic Element

Abstract

Cell-type-specific and inducible alternative splicing has a fundamental impact on regulating gene expression and cellular function in a variety of settings, including activation and differentiation. We have recently shown that activationinduced skipping of TRAF3 exon 8 activates noncanonical NF-B signaling upon T cell stimulation, but the regulatory basis for this splicing event remains unknown. Here we identify cis-and trans-regulatory elements rendering this splicing switch activation dependent and cell type specific. The cis-acting element is located 340 to 440 nucleotides upstream of the regulated exon and acts in a distance-dependent manner, since altering the location reduces its activity. A small interfering RNA screen, followed by cross-link immunoprecipitation and mutational analyses, identified CELF2 and hnRNP C as trans-acting factors that directly bind the regulatory sequence and together mediate increased exon skipping in activated T cells. CELF2 expression levels correlate with TRAF3 exon skipping in several model systems, suggesting that CELF2 is the decisive factor, with hnRNP C being necessary but not sufficient. These data suggest an interplay between CELF2 and hnRNP C as the mechanistic basis for activation-dependent alternative splicing of TRAF3 exon 8 and additional exons and uncover an intronic splicing silencer whose full activity depends on the precise location more than 300 nucleotides upstream of the regulated exon.KEYWORDS RNA binding proteins, RNA splicing S plicing-sensitive microarrays and transcriptome sequencing (RNA-Seq) technologies have led to the accumulation of enormous amounts of gene expression data from different cell types and tissues under various conditions. Such analyses have led to the conclusion that the majority of human genes are alternatively spliced and have shown strong differences in global splicing patterns under different conditions (1-3). Although these differences suggest a substantial contribution of alternative splicing to the regulation of gene expression, studies connecting alternative splicing and functionality are rare. Consequently, a functional impact of individual alternative splicing events on cellular functionality was demonstrated for only a few examples (4-7), and the function of most alternative splicing events remains unknown. However, a recent large-scale approach demonstrated that splice-isoforms of the same gene often have distinct protein-protein interaction profiles, further supporting the notion of distinct functionality (8).Another largely open question concerns splicing regulation: how are cell-typespecific splicing patterns established and maintained? Despite the identification of

The cancer-associated U2AF35 470A>G (Q157R) mutation creates an in-frame alternative 5′ splice site that impacts splicing regulation in Q157R patients

“…Q157P patients based on our subsequent analysis (see below; DLG1, FNTB, CEP57, DICER1, BCL2L12, and CHD3). In contrast to previous studies, we have used a well-established, radioactive RT-PCR protocol that allows a precise quantification of isoform ratios (Preußner et al 2014;Schultz et al 2016;Wilhelmi et al 2016; example in Fig. 2B).…”

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

“…S1C), which raises the possibility that the pathogenic missense variants might perturb these molecular features of CELF2 (Ladd and Cooper, 2004). To test this, we expressed wild-type (WT) and mutant CELF2 in human embryonic kidney (HEK) 293 cells and analyzed the alternative exon usage of known CELF2 splicing targets, PPP1R12A and TRAF3 (Schultz et al, 2017). CELF2 mutants induced exon skipping of both mRNAs to the same levels as wildtype (WT), which suggests that the pathogenic variants do not compromise the splicing activity of CELF2 (Fig.…”

The First Orally Deliverable Small Molecule for the Treatment of Spinal Muscular Atrophy

“…76 Similarly, Tra2-β1, a positive regulator of exon 7 splicing, turned out to be dispensable for inclusion of this exon in a Tra2-β1-deficient mouse model. 97,98 These observations are not entirely surprising given the redundancy and cross-regulation of splicing factors as, for example, observed for PTB, CELF2, and hnRNP C. 99-101…”

“…splicing, turned out to be dispensable for inclusion of this exon in a Tra2-β1-deficient mouse model. 97,98 These observations are not entirely surprising given the redundancy and crossregulation of splicing factors as, for example, observed for PTB, CELF2, and hnRNP C. [99][100][101] The discovery of the 15-nucleotide long ISS-N1 propelled the development of an antisense oligonucleotide (ASO)directed therapy for SMA. 89,102,103 Based on its strong inhibitory effect, ISS-N1 was dubbed as a master regulator of both splicing checkpoint and exon definition.…”