Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, limitations, and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for continuous extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies, while the containers and reproducible workflows could easily be deployed and extended to evaluate new methods or data sets.

Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation.

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.

Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing.

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.

Reciprocal regulation of hnRNP C and CELF2 through translation and transcription tunes splicing activity in T cells.

RNA binding proteins (RBPs) frequently regulate the expression of other RBPs in mammalian cells. Such cross-regulation has been proposed to be important to control networks of coordinated gene expression; however, much remains to be understood about how such networks of cross-regulation are established and what the functional consequence is of coordinated or reciprocal expression of RBPs. Here we demonstrate that the RBPs CELF2 and hnRNP C regulate the expression of each other, such that depletion of one results in reduced expression of the other. Specifically, we show that loss of hnRNP C reduces the transcription of CELF2 mRNA, while loss of CELF2 results in decreased efficiency of hnRNP C translation. We further demonstrate that this reciprocal regulation serves to fine tune the splicing patterns of many downstream target genes. Together, this work reveals new activities of hnRNP C and CELF2, provides insight into a previously unrecognized gene regulatory network, and demonstrates how cross-regulation of RBPs functions to shape the cellular transcriptome.

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes.

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.

Aberrant splicing in B-cell acute lymphoblastic leukemia.

Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing these samples to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded ∼100 SFs, e.g. hnRNPA1. HNRNPA1 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contributes to disease pathogenesis.

MAJIQ-SPEL: web-tool to interrogate classical and complex splicing variations from RNA-Seq data.

SUMMARY: Analysis of RNA sequencing (RNA-Seq) data have highlighted the fact that most genes undergo alternative splicing (AS) and that these patterns are tightly regulated. Many of these events are complex, resulting in numerous possible isoforms that quickly become difficult to visualize, interpret and experimentally validate. To address these challenges we developed MAJIQ-SPEL, a web-tool that takes as input local splicing variations (LSVs) quantified from RNA-Seq data and provides users with visualization and quantification of gene isoforms associated with those. Importantly, MAJIQ-SPEL is able to handle both classical (binary) and complex, non-binary, splicing variations. Using a matching primer design algorithm it also suggests to users possible primers for experimental validation by RT-PCR and displays those, along with the matching protein domains affected by the LSV, on UCSC Genome Browser for further downstream analysis. AVAILABILITY AND IMPLEMENTATION: Program and code will be available at http://majiq.biociphers.org/majiq-spel. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Integrative deep models for alternative splicing.

MOTIVATION: Advancements in sequencing technologies have highlighted the role of alternative splicing (AS) in increasing transcriptome complexity. This role of AS, combined with the relation of aberrant splicing to malignant states, motivated two streams of research, experimental and computational. The first involves a myriad of techniques such as RNA-Seq and CLIP-Seq to identify splicing regulators and their putative targets. The second involves probabilistic models, also known as splicing codes, which infer regulatory mechanisms and predict splicing outcome directly from genomic sequence. To date, these models have utilized only expression data. In this work, we address two related challenges: Can we improve on previous models for AS outcome prediction and can we integrate additional sources of data to improve predictions for AS regulatory factors. RESULTS: We perform a detailed comparison of two previous modeling approaches, Bayesian and Deep Neural networks, dissecting the confounding effects of datasets and target functions. We then develop a new target function for AS prediction in exon skipping events and show it significantly improves model accuracy. Next, we develop a modeling framework that leverages transfer learning to incorporate CLIP-Seq, knockdown and over expression experiments, which are inherently noisy and suffer from missing values. Using several datasets involving key splice factors in mouse brain, muscle and heart we demonstrate both the prediction improvements and biological insights offered by our new models. Overall, the framework we propose offers a scalable integrative solution to improve splicing code modeling as vast amounts of relevant genomic data become available. AVAILABILITY AND IMPLEMENTATION: Code and data available at: majiq.biociphers.org/jha_et_al_2017/. CONTACT: yosephb@upenn.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Induced transcription and stability of CELF2 mRNA drives widespread alternative splicing during T-cell 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 signal-induced 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.

Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells.

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.

In silico to in vivo splicing analysis using splicing code models.

With the growing appreciation of RNA splicing's role in gene regulation, development, and disease, researchers from diverse fields find themselves investigating exons of interest. Commonly, researchers are interested in knowing if an exon is alternatively spliced, if it is differentially included in specific tissues or in developmental stages, and what regulatory elements control its inclusion. An important step towards the ability to perform such analysis in silico was made with the development of computational splicing code models. Aimed as a practical how-to guide, we demonstrate how researchers can now use these code models to analyze a gene of interest, focusing on Bin1 as a case study. Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein known to be functionally regulated through alternative splicing in a tissue-specific manner. Specific Bin1 isoforms have been associated with muscular diseases and cancers, making the study of its splicing regulation of wide interest. Using AVISPA, a recently released web tool based on splicing code models, we show that many Bin1 tissue-dependent isoforms are correctly predicted, along with many of its known regulators. We review the best practices and constraints of using the tool, demonstrate how AVISPA is used to generate high confidence novel regulatory hypotheses, and experimentally validate predicted regulators of Bin1 alternative splicing.

Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4+ T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3'UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3'UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3'UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.

Alternative splicing of HDAC7 regulates its interaction with 14-3-3 proteins to alter histone marks and target gene expression.

Chromatin regulation and alternative splicing are both critical mechanisms guiding gene expression. Studies have demonstrated that histone modifications can influence alternative splicing decisions, but less is known about how alternative splicing may impact chromatin. Here, we demonstrate that several genes encoding histone-modifying enzymes are alternatively spliced downstream of T cell signaling pathways, including HDAC7, a gene previously implicated in controlling gene expression and differentiation in T cells. Using CRISPR-Cas9 gene editing and cDNA expression, we show that differential inclusion of HDAC7 exon 9 controls the interaction of HDAC7 with protein chaperones, resulting in changes to histone modifications and gene expression. Notably, the long isoform, which is induced by the RNA-binding protein CELF2, promotes expression of several critical T cell surface proteins including CD3, CD28, and CD69. Thus, we demonstrate that alternative splicing of HDAC7 has a global impact on histone modification and gene expression that contributes to T cell development.

RNA splicing analysis using heterogeneous and large RNA-seq datasets.

The ubiquity of RNA-seq has led to many methods that use RNA-seq data to analyze variations in RNA splicing. However, available methods are not well suited for handling heterogeneous and large datasets. Such datasets scale to thousands of samples across dozens of experimental conditions, exhibit increased variability compared to biological replicates, and involve thousands of unannotated splice variants resulting in increased transcriptome complexity. We describe here a suite of algorithms and tools implemented in the MAJIQ v2 package to address challenges in detection, quantification, and visualization of splicing variations from such datasets. Using both large scale synthetic data and GTEx v8 as benchmark datasets, we assess the advantages of MAJIQ v2 compared to existing methods. We then apply MAJIQ v2 package to analyze differential splicing across 2,335 samples from 13 brain subregions, demonstrating its ability to offer insights into brain subregion-specific splicing regulation.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, and limitations and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for seamless extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies. Furthermore, the containers and reproducible workflows generated in the course of this project can be seamlessly deployed and extended in the future to evaluate new methods or datasets.

Alternative splicing of apoptosis genes promotes human T cell survival.

Alternative splicing occurs in the vast majority of human genes, giving rise to distinct mRNA and protein isoforms. We, and others, have previously identified hundreds of genes that change their isoform expression upon T cell activation via alternative splicing; however, how these changes link activation input with functional output remains largely unknown. Here, we investigate how costimulation of T cells through the CD28 receptor impacts alternative splicing in T cells activated through the T cell receptor (TCR, CD3) and find that while CD28 signaling alone has minimal impact on splicing, it enhances the extent of change for up to 20% of TCR-induced alternative splicing events. Interestingly, a set of CD28-enhanced splicing events occur within genes encoding key components of the apoptotic signaling pathway; namely caspase-9, Bax, and Bim. Using both CRISPR-edited cells and antisense oligos to force expression of specific isoforms, we show for all three of these genes that the isoform induced by CD3/CD28 costimulation promotes resistance to apoptosis, and that changes in all three genes together function combinatorially to further promote cell viability. Finally, we show that the JNK signaling pathway, induced downstream of CD3/CD28 costimulation, is required for each of these splicing events, further highlighting their co-regulation. Together, these findings demonstrate that alternative splicing is a key mechanism by which costimulation of CD28 promotes viability of activated T cells.

Mutator dynamics in sexual and asexual experimental populations of yeast.

BACKGROUND: In asexual populations, mutators may be expected to hitchhike with associated beneficial mutations. In sexual populations, recombination is predicted to erode such associations, inhibiting mutator hitchhiking. To investigate the effect of recombination on mutators experimentally, we compared the frequency dynamics of a mutator allele (msh2Δ) in sexual and asexual populations of Saccharomyces cerevisiae. RESULTS: Mutator strains increased in frequency at the expense of wild-type strains in all asexual diploid populations, with some approaching fixation in 150 generations of propagation. Over the same period of time, mutators declined toward loss in all corresponding sexual diploid populations as well as in haploid populations propagated asexually. CONCLUSIONS: We report the first experimental investigation of mutator dynamics in sexual populations. We show that a strong mutator quickly declines in sexual populations while hitchhiking to high frequency in asexual diploid populations, as predicted by theory. We also show that the msh2Δ mutator has a high and immediate realized cost that is alone sufficient to explain its decline in sexual populations. We postulate that this cost is indirect; namely, that it is due to a very high rate of recessive lethal or strongly deleterious mutation. However, we cannot rule out the possibility that msh2Δ also has unknown directly deleterious effects on fitness, and that these effects may differ between haploid asexual and sexual populations. Despite these reservations, our results prompt us to speculate that the short-term cost of highly deleterious recessive mutations can be as important as recombination in preventing mutator hitchhiking in sexual populations.

Analysis of scientific society honors reveals disparities.

Delivering a keynote talk at a conference organized by a scientific society or being named as a fellow by such a society indicates that a scientist is held in high regard by their colleagues. To explore if the distribution of such indicators of esteem in the field of bioinformatics reflects the composition of this field, we compared the gender, name origin, and country of affiliation of 412 honorees from the "International Society for Computational Biology" (75 fellows and 337 keynote speakers) with over 170,000 last authorships on computational biology papers between 1993 and 2019. The proportion of honors bestowed on women was similar to that of the field's overall last authorship rate. However, names of East Asian origin have been persistently underrepresented among honorees. Moreover, there were roughly twice as many honors bestowed on scientists with an affiliation in the United States as expected based on literature authorship. A record of this paper's transparent peer review process is included in the supplemental information.

CYP11B1 variants influence skeletal maturation via alternative splicing.

We performed genome-wide association study meta-analysis to identify genetic determinants of skeletal age (SA) deviating in multiple growth disorders. The joint meta-analysis (N = 4557) in two multiethnic cohorts of school-aged children identified one locus, CYP11B1 (expression confined to the adrenal gland), robustly associated with SA (rs6471570-A; ß = 0.14; P = 6.2 × 10-12). rs6410 (a synonymous variant in the first exon of CYP11B1 in high LD with rs6471570), was prioritized for functional follow-up being second most significant and the one closest to the first intron-exon boundary. In 208 adrenal RNA-seq samples from GTEx, C-allele of rs6410 was associated with intron 3 retention (P = 8.11 × 10-40), exon 4 inclusion (P = 4.29 × 10-34), and decreased exon 3 and 5 splicing (P = 7.85 × 10-43), replicated using RT-PCR in 15 adrenal samples. As CYP11B1 encodes 11-ß-hydroxylase, involved in adrenal glucocorticoid and mineralocorticoid biosynthesis, our findings highlight the role of adrenal steroidogenesis in SA in healthy children, suggesting alternative splicing as a likely underlying mechanism.