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Significance Alternative pre-mRNA splicing is a regulated process that greatly diversifies gene products by changing the exons incorporated into mRNA. This process is dysregulated in cancers. Here, we studied exon usage in aggressive prostate cancers and linked exon incorporation decisions to cancer driver genes. Through computational and experimental studies, we found that a strong cancer driver gene, Myc, was linked to exon changes in genes that themselves regulate alternative splicing. These exons often encoded premature stop codons that would decrease gene expression, suggestive of a Myc-driven autoregulatory loop to help control levels of splicing regulatory proteins., We sought to define the landscape of alternative pre-mRNA splicing in prostate cancers and the relationship of exon choice to known cancer driver alterations. To do so, we compiled a metadataset composed of 876 RNA-sequencing (RNA-Seq) samples from five publicly available sources representing a range of prostate phenotypes from normal tissue to drug-resistant metastases. We subjected these samples to exon-level analysis with rMATS-turbo, purpose-built software designed for large-scale analyses of splicing, and identified 13,149 high-confidence cassette exon events with variable incorporation across samples. We then developed a computational framework, pathway enrichment-guided activity study of alternative splicing (PEGASAS), to correlate transcriptional signatures of 50 different cancer driver pathways with these alternative splicing events. We discovered that Myc signaling was correlated with incorporation of a set of 1,039 cassette exons enriched in genes encoding RNA binding proteins. Using a human prostate epithelial transformation assay, we confirmed the Myc regulation of 147 of these exons, many of which introduced frameshifts or encoded premature stop codons. Our results connect changes in alternative pre-mRNA splicing to oncogenic alterations common in prostate and many other cancers. We also establish a role for Myc in regulating RNA splicing by controlling the incorporation of nonsense-mediated decay-determinant exons in genes encoding RNA binding proteins.

Rbfox proteins control alternative splicing and posttranscriptional regulation in mammalian brain and are implicated in neurological disease. These proteins recognize the RNA sequence (U)GCAUG, but their structures and diverse roles imply a variety of protein-protein interactions. We find that nuclear Rbfox proteins are bound within a large assembly of splicing regulators (LASR), a multimeric complex containing the proteins hnRNP M, hnRNP H, hnRNP C, Matrin3, NF110/NFAR-2, NF45, and DDX5, all approximately equimolar to Rbfox. We show that splicing repression mediated by hnRNP M is stimulated by Rbfox. Virtually all the intron-bound Rbfox is associated with LASR, and hnRNP M motifs are enriched adjacent toRbfox crosslinking sites invivo. These findings demonstrate that Rbfox proteins bind RNA with a defined set of cofactors and affect a broader set ofexons than previously recognized. The function of this multimeric LASR complex has implications for deciphering the regulatory codes controlling splicing networks.

Summary Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3ʹ UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease. Video Abstract

We show that the splicing regulator PTBP2 controls a genetic program essential for neuronal maturation. Depletion of PTBP2 in developing mouse cortex leads to degeneration of these tissues over the first three postnatal weeks, a time when the normal cortex expands and develops mature circuits. Cultured Ptbp2−/− neurons exhibit the same initial viability as wild type, with proper neurite outgrowth and marker expression. However, these mutant cells subsequently fail to mature and die after a week in culture. Transcriptome-wide analyses identify many exons that share a pattern of mis-regulation in the mutant brains, where isoforms normally found in adults are precociously expressed in the developing embryo. These transcripts encode proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program, where PTBP2 acts to temporarily repress expression of adult protein isoforms until the final maturation of the neuron. DOI: http://dx.doi.org/10.7554/eLife.01201.001, eLife digest Cells within the developing brain undergo an extended period of maturation. A neuronal progenitor cell must first migrate to the proper place within the brain and then develop long extensions that become the axon and dendrites used by the mature neuron to communicate with other cells. Finally, the synapses that connect neurons with other neurons must be established. Multiple mechanisms are needed to ensure that all the proteins involved in this process are expressed when and where they are needed. The production of a protein begins with a region of DNA being transcribed to produce an RNA transcript that consists of segments called exons separated by segments called introns. This transcript then undergoes a process called splicing that involves the introns being removed and the exons being joined together to form a messenger RNA molecule that can be translated into protein. Specialized RNA binding proteins regulate the splicing process, and most RNA transcripts are subject to a form of splicing called alternative splicing that allows a single gene to express more than one messenger RNA molecule and hence more than one protein product. As neuronal progenitor cells in the brain are induced to mature into neurons, many RNA transcripts are seen to change their splicing patterns. At the same time, the level of a regulatory RNA binding protein called PTBP1 decreases and the level of a related protein called PTBP2 increases. Now Li et al. have studied mutant mice that lack PTBP2, and have found that structures of the forebrain that normally undergo extensive development after birth instead experience tissue degeneration when PTBP2 is absent. Similarly, when neurons lacking PTPB2 are grown in culture, they fail to develop correctly and die. Li et al. also found that messenger RNAs from many genes involved in postnatal brain development—affecting processes such as the growth of axons and dendrites and the formation of synapses—exhibit defective alternative splicing in the mutant mice. Specifically, protein variants that would normally be expressed only in adult brains were being expressed much earlier. By inhibiting the expression of adult forms of proteins until neurons have matured, PTBP2 plays an essential role in controlling the brain’s early development. Further work is now required to determine how individual changes in messenger RNA and protein structure controlled by PTBP2 might alter protein function between immature and mature neurons. DOI: http://dx.doi.org/10.7554/eLife.01201.002

Proteins of the Rbfox family act with a complex ofproteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and thisdomain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.

The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development. DOI: http://dx.doi.org/10.7554/eLife.09268.001, eLife digest The neurons that transmit information around the nervous system develop in several stages. Embryonic stem cells specialize to form neuronal progenitor cells, which then develop into neurons. These cell types have different characteristics, in part because they make different proteins or different versions of the same proteins. To make a protein, the DNA sequence of a gene is used to build a molecule of ribonucleic acid (RNA) that acts as a template for the protein. However, not all of this sequence codes for the protein. The non-coding regions must be removed from the RNA, and the remaining “exons” joined together to form the final “mRNA” template. Not all of the exons are necessarily included in the final mRNA molecule. By joining together different combinations of exons, several different versions of a protein can be produced from a single gene. This process is known as alternative splicing. One way that alternative splicing is controlled is through proteins that bind to RNA and determine which exons are included or excluded from the final mRNA molecule. PTBP1 is an RNA-binding protein that controls alternative splicing in embryonic stem cells and neuronal progenitor cells. Embryonic stem cells have the ability to develop into all the cells of the body. In contrast, neuronal progenitor cells are restricted in their development and only give rise to specialized cells of the nervous system. The role of PTBP1 in these properties was not clear. Linares et al. have now used a range of techniques to study the RNA molecules produced in these two cell types and how these RNAs change when PTBP1 is removed. This identified many RNAs whose splicing is regulated by PTBP1, including mRNAs of the gene that produces a protein called Pbx1, which is an important regulator of neuronal development. Further investigation revealed that PTBP1 prevents a particular exon being included in the mRNA template for Pbx1. This creates an embryonic stem cell form of Pbx1 that does not affect neuronal genes. Removal of PTBP1 allows splicing of the Pbx1 exon and produces a version of Pbx1 that is found in neuronal progenitor cells and which turns on neuronal genes. Thus, through its action on Pbx1, one role of PTBP1 is to enable stem cells to maintain their non-neuronal properties and prevent their premature development into neuronal progenitor cells. The gene for Pbx1 is only one of many genes controlled by PTBP1 at the level of splicing. One challenge for the future will be to understand how these genes work together in a common program that determines the properties of stem cells. Another question regards how the different Pbx1 proteins in stem cells and in neuronal progenitors can exert different effects in the cells where they are made. DOI: http://dx.doi.org/10.7554/eLife.09268.002

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.

Alternative splicing has emerged as a promising therapeutic target in a number of human disorders. However, the discovery of compounds that target the splicing reaction has been hindered by the lack of suitable high-throughput screening assays. Conversely, the effects of known drugs on the splicing reaction are mostly unclear and not routinely assessed. We have developed a two-color fluorescent reporter for cellular assays of exon inclusion that can accommodate nearly any cassette exon and minimizes interfering effects from changes in transcription and translation. We used microtubule-associated protein tau (MAPT) exon 10, whose missplicing causes frontotemporal dementia, to test the reporter in screening libraries of known bioactive compounds. These screens yielded several compounds that alter the splicing of the exon, both in the reporter and in the endogenous MAPT mRNA. One compound, digoxin, has long been used in the treatment of heart failure, but was not known to modulate splicing. The positive compounds target different signal transduction pathways, and microarray analysis shows that each compound affects the splicing of a different set of exons in addition to MAPT exon 10. Our results identify currently prescribed cardiotonic steroids as modulators of alternative splicing and demonstrate the feasibility of screening for drugs that alter exon inclusion.

Many metazoan gene transcripts exhibit neuron-specific splicing patterns, but the developmental control of these splicing events is poorly understood. We show that the splicing of a large group of exons is reprogrammed during neuronal development by a switch in expression between two highly similar polypyrimidine tract-binding proteins, PTB and nPTB (neural PTB). PTB is a well-studied regulator of alternative splicing, but nPTB is a closely related paralog whose functional relationship to PTB is unknown. In the brain, nPTB protein is specifically expressed in post-mitotic neurons, whereas PTB is restricted to neuronal precursor cells (NPC), glia, and other nonneuronal cells. Interestingly, nPTB mRNA transcripts are found in NPCs and other nonneuronal cells, but in these cells nPTB protein expression is repressed. This repression is due in part to PTB-induced alternative splicing of nPTB mRNA, leading to nonsense-mediated decay (NMD). However, we find that even properly spliced mRNA fails to express nPTB protein when PTB is present, indicating contributions from additional post-transcriptional mechanisms. The PTB-controlled repression of nPTB results in a mutually exclusive pattern of expression in the brain, where the loss of PTB in maturing neurons allows the synthesis of nPTB in these cells. To examine the consequences of this switch, we used splicing-sensitive microarrays to identify different sets of exons regulated by PTB, nPTB, or both proteins. During neuronal differentiation, the splicing of these exon sets is altered as predicted from the observed changes in PTB and nPTB expression. These data show that the post-transcriptional switch from PTB to nPTB controls a widespread alternative splicing program during neuronal development.

We show that the splicing regulator PTBP2 controls a genetic program essential for neuronal maturation. Depletion of PTBP2 in developing mouse cortex leads to degeneration of these tissues over the first three postnatal weeks, a time when the normal cortex expands and develops mature circuits. Cultured Ptbp2(-/-) neurons exhibit the same initial viability as wild type, with proper neurite outgrowth and marker expression. However, these mutant cells subsequently fail to mature and die after a week in culture. Transcriptome-wide analyses identify many exons that share a pattern of mis-regulation in the mutant brains, where isoforms normally found in adults are precociously expressed in the developing embryo. These transcripts encode proteins affecting neurite growth, pre- and post-synaptic assembly, and synaptic transmission. Our results define a new genetic regulatory program, where PTBP2 acts to temporarily repress expression of adult protein isoforms until the final maturation of the neuron. DOI: http://dx.doi.org/10.7554/eLife.01201.001.

The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3′ end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3′-processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts but their role in neuronal physiology is not clear1. We show here that central nervous system (CNS)-specific deletion of the Rbfox1 gene results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording reveals a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole transcriptome analyses identify multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation, some of which are implicated in human epilepsy. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function.