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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 frontotemporat 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. [ABSTRACT FROM AUTHOR]

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, purposebuilt 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 premRNA 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. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem- loop to inhibit mature miR-124 expression in vivo and DROSHA cleavage of pri-miR-124-1 in vitro. This function for PTBP1 in repressing miR-124 biogenesis defines an additional regulatory loop in the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often found between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct derepression of miR-124, in addition to indirect effects previously described. [ABSTRACT FROM AUTHOR]

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts, but its role in neuronal physiology is not clear. We show here that central nervous system-specific deletion of the gene encoding Rbfox1 results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording revealed a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole-transcriptome analyses identified 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]