The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor–armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. Significance CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Alternative splicing (AS) can critically affect gene function and disease, yet mapping splicing variations remains a challenge. Here, we propose a new approach to define and quantify mRNA splicing in units of local splicing variations (LSVs). LSVs capture previously defined types of alternative splicing as well as more complex transcript variations. Building the first genome wide map of LSVs from twelve mouse tissues, we find complex LSVs constitute over 30% of tissue dependent transcript variations and affect specific protein families. We show the prevalence of complex LSVs is conserved in humans and identify hundreds of LSVs that are specific to brain subregions or altered in Alzheimer's patients. Amongst those are novel isoforms in the Camk2 family and a novel poison exon in Ptbp1, a key splice factor in neurogenesis. We anticipate the approach presented here will advance the ability to relate tissue-specific splice variation to genetic variation, phenotype, and disease.DOI: http://dx.doi.org/10.7554/eLife.11752.001

Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ∼25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling.

Studies in several cell types have highlighted dramatic and diverse changes in mRNA processing that occur upon cellular stimulation. However, the mechanisms and pathways that lead to regulated changes in mRNA processing remain poorly understood. Here we demonstrate that expression of the splicing factor CELF2 (CUGBP, Elav-like family member 2) is regulated in response to T-cell signaling through combined increases in transcription and mRNA stability. Transcriptional induction occurs within 6 h of stimulation and is dependent on activation of NF-κB. Subsequently, there is an increase in the stability of the CELF2 mRNA that correlates with a change in CELF2 3′UTR length and contributes to the total signalinduced enhancement of CELF2 expression. Importantly, we uncover dozens of splicing events in cultured T cells whose changes upon stimulation are dependent on CELF2 expression, and provide evidence that CELF2 controls a similar proportion of splicing events during human thymic T-cell development. Taken together, these findings expand the physiologic impact of CELF2 beyond that previously documented in developing neuronal and muscle cells to T-cell development and function, identify unappreciated instances of alternative splicing in the human thymus, and uncover novel mechanisms for CELF2 regulation that may broadly impact CELF2 expression across diverse cell types.alternative splicing | CELF2 | thymic development | T cells | mRNA stability

CELF2 is an RNA binding protein that has been implicated in developmental and signal-dependent splicing in the heart, brain and T cells. In the heart, CELF2 expression decreases during development, while in T cells CELF2 expression increases both during development and in response to antigen-induced signaling events. Although hundreds of CELF2-responsive splicing events have been identified in both heart and T cells, the way in which CELF2 functions has not been broadly investigated. Here we use CLIP-Seq to identified physical targets of CELF2 in a cultured human T cell line. By comparing the results with known functional targets of CELF2 splicing regulation from the same cell line we demonstrate a generalizable position-dependence of CELF2 activity that is consistent with previous mechanistic studies of individual CELF2 target genes in heart and brain. Strikingly, this general position-dependence is sufficient to explain the bi-directional activity of CELF2 on 2 T cell targets recently reported. Therefore, we propose that the location of CELF2 binding around an exon is a primary predictor of CELF2 function in a broad range of cellular contexts.

Three of the eight RNA segments encoded by the influenza A virus (IAV) undergo alternative splicing to generate distinct proteins. Previously, we found that host proteins hnRNP K and NS1-BP regulate IAV M segment splicing, but the mechanistic details were unknown. Here we show NS1-BP and hnRNP K bind M mRNA downstream of the M2 5′ splice site (5′ss). NS1-BP binds most proximal to the 5′ss, partially overlapping the U1 snRNP binding site, while hnRNP K binds further downstream and promotes U1 snRNP recruitment. Mutation of either or both the hnRNP K and NS1-BP-binding sites results in M segment mis-splicing and attenuated IAV replication. Additionally, we show that hnRNP K and NS1-BP regulate host splicing events and that viral infection causes mis-splicing of some of these transcripts. Therefore, our proposed mechanism of hnRNP K/NS1-BP mediated IAV M splicing provides potential targets of antiviral intervention and reveals novel host functions for these proteins.

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 coregulation 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 coregulated 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, Drosophila, and Caenorhabditis elegans suggests this antagonism is conserved throughout evolution.

Every successful cancer therapy story has Exhibit B, comprised of patients who either did not respond to the initial treatment or acquired resistance after a seemingly curative intervention. The CD19-directed chimeric antigen receptorarmed T-cell therapy (commonly known as CART-19) is the case in point. Although it has revolutionized treatment for B-cell acute lymphoblastic leukemia (B-ALL) in children and adults and gained swift FDA approval,~30% of