While the use of next-generation androgen receptor pathway inhibition (ARPI) therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC), several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR) indifferent and gain a neuroendocrine (NE)-like phenotype. This subtype of castration-resistant prostate cancer has been termed "treatment-induced castration-resistant neuroendocrine prostate cancer" (CRPC-NE). Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4), a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4 and SRRM4-mediated splicing in driving a CRPC-NE program as a coping mechanism for therapy resistance, as well as define future SRRM4-targeted therapeutic approaches for treating CRPC-NE or mitigating its development.

Neuroendocrine prostate cancer (NEPC) is becoming more prevalent as more potent androgen receptor (AR) pathway inhibitors are applied to patients with metastatic tumors. However, there are limited cell and xenograft models currently available, hindering the investigation of signal pathways involved in regulating NEPC progression and the design of high throughput screening assays for inhibitors to treat NEPC patients. Here, we report an NEPC model, LnNE, that is derived from prostate adenocarcinoma cells and has global similarity in transcription and RNA splicing to tumors from NEPC patients. LnNE xenografts are castrate-resistant and highly aggressive. Its tumor take is ∼3-5 weeks and tumor doubling time is ∼2-3 weeks. LnNE expresses multiple neuroendocrine markers, preserves AR expression, but is PSA negative. Its neuroendocrine phenotype cannot be reversed by androgen treatment. LnNE cells grow as multi-cellular spheroids under 2-dimensional culture conditions similar to the NEPC cell line NCI-H660, but have higher proliferation rate and are easier to be transfected. LnNE cells can also adapt to 3-dimensional culture conditions in a 96-plate format, allowing high throughput screening assays. In summary, the LnNE model is useful to study the mechanisms of NEPC progression and to discover potential therapies for NEPC.

In mechanosensory hair cells (HCs) of the ear, the transcriptional repressor REST is continuously inactivated by alternative splicing of its pre-mRNA. This mechanism of REST inactivation is crucial for hearing in humans and mice. Rest is one of many pre-mRNAs whose alternative splicing is regulated by the splicing factor SRRM4; Srrm4 loss-of-function mutation in mice (Srrm4 ^bv/bv ) causes deafness, balance defects, and degeneration of all HC types other than the outer HCs (OHCs). The specific splicing alterations that drive HC degeneration in Srrm4 ^bv/bv mice are unknown, and the mechanism underlying SRRM4-independent survival of OHCs is undefined. Here, we show that transgenic expression of a dominant-negative REST fragment in Srrm4 ^bv/bv mice is sufficient for long-term rescue of hearing, balancing, HCs, alternative splicing of Rest, and expression of REST target genes including the Srrm4 paralog Srrm3 We also show that in HCs, SRRM3 regulates many of the same exons as SRRM4; OHCs are unique among HCs in that they transiently down-regulate Rest transcription as they mature to express Srrm3 independently of SRRM4; and simultaneous SRRM4-SRRM3 deficiency causes complete HC loss by preventing inactivation of REST in all HCs. Thus, our data reveal that REST inactivation is the primary and essential role of SRRM4 in the ear, and that OHCs differ from other HCs in the SRRM4-independent expression of the functionally SRRM4-like splicing factor SRRM3.

Potent androgen receptor pathway inhibition (ARPI) therapies has given rise to a lethal, aggressive subtype of castration-resistant prostate cancer (CRPC) called treatment-induced neuroendocrine prostate cancer (t-NEPC). Now, t-NEPC poses a major clinical issue as ~20% of CRPC cases bear this subtype-a rate of occurrence that is predicted to rise with the widespread use of ARPI therapies. Unfortunately, there are no targeted therapies currently available to treat t-NEPC, as the origin and molecular underpinnings of t-NEPC development remain unclear. In this study, we identify that the RNA splicing of the G protein-coupled receptor kinase-interacting protein 1 (GIT1) gene is a unique event in t-NEPC patients. Specifically, the up-regulation of the GIT1-A splice variant and down-regulation of the GIT1-C variant are associated with t-NEPC progression in patient tumors, as well as mouse and cell models, when compared to expression in the prostate adenocarcinoma subtype. RNA-binding assays reveal that the RNA splicing of GIT1 is directly driven by SRRM4 and is associated with its expression in CRPC cohorts. We show that GIT1-A and GIT1-C regulate differential transcriptomes in prostate cancer cells, where GIT1-A is enriched with genes associated with cell morphogenesis and neural functions. In summary, our study is the first to report that alternative RNA splicing of the GIT1 gene is associated with t-NEPC and may regulate transcriptomes implicated in t-NEPC progression. This article is protected by copyright. All rights reserved.

The neural tube is the primordium of the central nervous system (CNS) in which its development is not entirely clear. Understanding the cellular and molecular basis of neural tube development could, therefore, provide vital clues to the mechanism of neural tube defects (NTDs). Here, we investigated the gene expression profiles of three different time points (embryonic day (E) 8.5, 9.5 and 10.5) of mouse neural tube by using RNA-seq approach. About 391 differentially expressed genes (DEGs) were screened during mouse neural tube development, including 45 DEGs involved in CNS development, among which Bmp2, Ascl1, Olig2, Lhx1, Wnt7b and Eomes might play the important roles. Of 45 DEGs, Foxp2, Eomes, Hoxb3, Gpr56, Hap1, Nkx2-1, Sez6l2, Wnt7b, Tbx20, Nfib, Cntn1 and Dcx had different isoforms, and the opposite expression pattern of different isoforms was observed for Gpr56, Nkx2-1 and Sez6l2. In addition, alternative splicing, such as mutually exclusive exon, retained intron, skipped exon and alternative 3' splice site was identified in 10 neural related differentially splicing genes, including Ngrn, Ddr1, Dctn1, Dnmt3b, Ect2, Map2, Mbnl1, Meis2, Vcan and App. Moreover, seven neural splicing factors, such as Nova1/2, nSR100/Srrm4, Elavl3/4, Celf3 and Rbfox1 were differentially expressed during mouse neural tube development. Interestingly, nine DEGs identified above were dysregulated in retinoic acid-induced NTDs model, indicating the possible important role of these genes in NTDs. Taken together, our study provides more comprehensive information on mouse neural tube development, which might provide new insights on NTDs occurrence. © 2017 IUBMB Life, 69(9):706-719, 2017.

Loss of a cell's ability to terminally differentiate because of mutations is a selected genetic event in tumorigenesis. Genomic analyses of low-grade glioma have reported recurrent mutations of far upstream element-binding protein 1 (FUBP1). Here, we show that FUBP1 expression is dynamically regulated during neurogenesis and that its downregulation in neural progenitors impairs terminal differentiation and promotes tumorigenesis collaboratively with expression of IDH1R132H. Mechanistically, collaborative action between SRRM4 and FUBP1 is necessary for mini-exon splicing of the neurospecific LSD1+8a isoform. LSD1+8a was downregulated upon loss of FUBP1 in neural progenitors, thereby impairing terminal neuronal differentiation and maturation. Reinforcing LSD1+8a expression in FUBP1-downregulated neural progenitors restored terminal differentiation and suppressed tumorigenesis; hence, LSD1+8a is an obligatory effector of FUBP1-dependent neuronal differentiation. These findings establish a direct role for FUBP1 in neuronal differentiation and also explain its tumor-suppressor function in the nervous system.

Taking advantage of the fast-growing knowledge of RNA-binding proteins (RBPs) we review the signature of downregulated genes for RBPs in the transcriptome of induced pluripotent stem cell neurons (iNeurons) modelling the neurodevelopmental Rubinstein Taybi Syndrome (RSTS) caused by mutations in the genes encoding CBP/p300 acetyltransferases. We discuss top and functionally connected downregulated genes sorted to "RNA processing" and "Ribonucleoprotein complex biogenesis" Gene Ontology clusters. The first set of downregulated RBPs includes members of hnRNHP (A1, A2B1, D, G, H2-H1, MAGOHB, PAPBC), core subunits of U small nuclear ribonucleoproteins and Serine-Arginine splicing regulators families, acting in precursor messenger RNA alternative splicing and processing. Consistent with literature findings on reduced transcript levels of serine/arginine repetitive matrix 4 (SRRM4) protein, the main regulator of the neural-specific microexons splicing program upon depletion of Ep300 and Crebbp in mouse neurons, RSTS iNeurons show downregulated genes for proteins impacting this network. We link downregulated genes to neurological disorders including the new HNRNPH1-related intellectual disability syndrome with clinical overlap to RSTS. The set of downregulated genes for Ribosome biogenesis includes several components of ribosomal subunits and nucleolar proteins, such NOP58 and fibrillarin that form complexes with snoRNAs with a central role in guiding post-transcriptional modifications needed for rRNA maturation. These nucleolar proteins are "dual" players as fibrillarin is also required for epigenetic regulation of ribosomal genes and conversely NOP58-associated snoRNA levels are under the control of NOP58 interactor BMAL1, a transcriptional regulator of the circadian rhythm. Additional downregulated genes for "dual specificity" RBPs such as RUVBL1 and METTL1 highlight the links between chromatin and the RBP-ome and the contribution of perturbations in their cross-talk to RSTS. We underline the hub position of CBP/p300 in chromatin regulation, the impact of its defect on neurons' post-transcriptional regulation of gene expression and the potential use of epidrugs in therapeutics of RBP-caused neurodevelopmental disorders.

Keywords: CBP/p300; RNA-binding proteins; RNASeq; Rubinstein-Taybi; alternative splicing; chromatin regulators; downregulated genes; induced pluripotent stem cell-neurons; neurodevelopmental disorders; ribosome biogenesis.

Alternative splicing (AS) is a posttranscriptional mechanism regulating gene expression that complex organisms utilize to expand proteome diversity from a comparatively limited set of genes. Recent research has increasingly associated AS with increased functional complexity in the central nervous systems in higher order mammals. This work has heavily implicated aberrant AS in several neurocognitive and neurodevelopmental disorders, including autism. Due to the strong genetic association between germline PTEN mutations and autism spectrum disorder (ASD), we hypothesized that germline PTEN mutations would alter AS patterns, contributing to the pathophysiology of ASD. In a murine model of constitutional mislocalization of Pten, recapitulating an autism-like phenotype, we found significant changes in AS patterns across the neural transcriptome by analyzing RNA-sequencing data with the program rMATS. A few hundred significant alternative splicing events (ASEs) that differentiate each m3m4 genotype were identified. These ASEs occur in genes enriched in PTEN signaling, inositol metabolism, and several other pathways relevant to the pathophysiology of ASD. In addition, we identified expression changes in several splicing factors known to be enriched in the nervous system. For instance, the master regulator of microexons, Srrm4, has decreased expression, and consequently, we found decreased inclusion of microexons in the Pten^m3m4/m3m4 cortex (~10% decrease). We also demonstrated that the m3m4 mutation disrupts the interaction between Pten and U2af2, a member of the spliceosome. In sum, our observations point to germline Pten disruption changing the landscape of alternative splicing in the brain, and these changes may be relevant to the pathogenesis and/or maintenance of PTEN-ASD phenotypes.

Small cell lung cancer (SCLC) is the most aggressive neuroendocrine phenotype of the deadliest human lung cancers. However the therapeutic landscape for SCLC has not changed in over 30 years. Effective treatment and prognosis are needed to combat this aggressive cancer. Herein we report that Ser/Arg repetitive matrix 4 (SRRM4), a splicing activator, is abnormally expressed at high levels in SCLC and thus is a potential therapeutic target. We screened an effective gapmer antisense oligonucleotide (gASO) targeting SRRM4 in vitro which led to cell death of SCLC. Our gASO, which is stabilized by containing artificial nucleotides, effectively represses SRRM4 mRNA. We found that our gASO repressed SRRM4 synthesis leading to a dramatic tumor reduction in a lung cancer mouse model. We also analyzed miRNA microarray and found that the miR-4516 is abnormally increased in exosomes in the blood of SCLC patients. Treating with gASO suppressed tumors in the SCLC model mouse concurrently reduced plasma miR-4516. In conclusion this study reports that administration of an SRRM4-targeted gASO coupled with a novel miRNA diagnostic methodology represents a potential breakthrough in the therapeutic treatment of high mortality SCLC.

Dysfunctional islets of Langerhans are a hallmark of type 2 diabetes (T2D). We hypothesize that differences in islet gene expression alternative splicing which can contribute to altered protein function also participate in islet dysfunction. RNA sequencing (RNAseq) data from islets of obese diabetes-resistant and diabetes-susceptible mice were analyzed for alternative splicing and its putative genetic and epigenetic modulators. We focused on the expression levels of chromatin modifiers and SNPs in regulatory sequences. We identified alternative splicing events in islets of diabetes-susceptible mice amongst others in genes linked to insulin secretion, endocytosis or ubiquitin-mediated proteolysis pathways. The expression pattern of 54 histones and chromatin modifiers, which may modulate splicing, were markedly downregulated in islets of diabetic animals. Furthermore, diabetes-susceptible mice carry SNPs in RNA-binding protein motifs and in splice sites potentially responsible for alternative splicing events. They also exhibit a larger exon skipping rate, e.g., in the diabetes gene Abcc8, which might affect protein function. Expression of the neuronal splicing factor Srrm4 which mediates inclusion of microexons in mRNA transcripts was markedly lower in islets of diabetes-prone compared to diabetes-resistant mice, correlating with a preferential skipping of SRRM4 target exons. The repression of Srrm4 expression is presumably mediated via a higher expression of miR-326-3p and miR-3547-3p in islets of diabetic mice. Thus, our study suggests that an altered splicing pattern in islets of diabetes-susceptible mice may contribute to an elevated T2D risk.

Keywords: MicroRNA; RNAseq; alternative splicing; diabetes; epigenetic; β-cell failure.

Objective: To explore the differences in peripheral blood markers between HIV well-controlled patients on long-term suppressive antiretroviral therapy (HIV-group) and age-matched healthy controls (HC), to evaluate the benefits of virological suppression in those patients.

Methods: We performed a case-control study in 22 subjects in HIV-group and 14 in HC-group. RNA-seq analysis was performed from peripheral blood mononuclear cells (PBMCs). Peripheral blood T-cell subsets were evaluated by flow cytometry and plasma biomarkers by immunoassays. All p-values were corrected by the false discovery rate (FDR, q-values).

Results: Only the serine/arginine repetitive matrix 4 (SRRM4) gene, which is involved in alternative RNA splicing events, was differentially expressed between HIV and HC groups (q-value≤0.05 and fold-change≥2). However, 147 differentially expressed genes were found with a more relaxed threshold (p-value≤0.05 and fold-change≥1.5), of which 67 genes with values of variable importance in projection (VIP)≥1 were selected for pathway analysis. We found that six ribosomal genes represented significant Ribosome-related pathways, all of them downregulated in the HIV-group, which may be a strategy to facilitate viral production. T-cells subset and plasma biomarkers did not show significant differences after FDR correction (q-value> 0.05), but a non-corrected analysis showed higher values of CD4 Treg cells (CD4CD25CD127), MCP-1, and sVEGF-R1 in the HIV-group (p-value≤0.05).

Conclusions: T-cell subsets, plasma biomarkers, and gene expression were close to normalization in HIV-infected patients on long-term suppressive cART compared to healthy controls. However, residual alterations remain, mainly at the gene expression, which still reveals the impact of HIV infection in these patients.

High-throughput RNA sequencing (RNA-seq) and dedicated bioinformatics pipelines have synergized to identify an expansive repertoire of unique circular RNAs (circRNAs), exceeding 100,000 variants. While the vast majority of these circRNAs comprise canonical exonic and intronic sequences, microexons (MEs)-which occur in 30% of functional mRNA transcripts-have been entirely overlooked. CircRNAs which contain these known MEs (ME-circRNAs) could be identified with commonly utilized circRNA prediction pipelines, CIRCexplorer2 and CIRI2, but were not previously recognized as ME-circRNAs. In addition, when employing a bespoke bioinformatics pipeline for identifying RNA chimeras, called Hyb, we could also identify over 2000 ME-circRNAs which contain novel MEs at their backsplice junctions, that are uncalled by either CIRCexplorer2 or CIRI2. Analysis of circRNA-seq datasets from gliomas of varying clinical grades compared with matched control tissue has shown circRNAs have potential as prognostic markers for stratifying tumor from healthy tissue. Furthermore, the abundance of microexon-containing circRNAs (ME-circRNAs) between tumor and normal tissues is correlated with the expression of a splicing associated factor, Serine/arginine repetitive matrix 4 (SRRM4). Overexpressing SRRM4, known for regulating ME inclusion in mRNAs critical for neural differentiation, in human HEK293 cells resulted in the biogenesis of over 2000 novel ME-circRNAs, including ME-circEIF4G3, and changes in the abundance of many canonical circRNAs, including circSETDB2 and circLBRA. This shows SRRM4, in which its expression is correlated with poor prognosis in gliomas, acts as a bona fide circRNA biogenesis factor. Given the known roles of MEs and circRNAs in oncogenesis, the identification of these previously unrecognized ME-circRNAs further increases the complexity and functional purview of this non-coding RNA family.

Keywords: SRRM4; alternative splicing; circular RNAs; glioblastoma; microexons; splicing factors.

The diagnosis and management of concussion is hindered by its diverse clinical presentation and assessment tools reliant upon subjectively-experienced symptoms. The biomechanical threshold of concussion is also not well understood and asymptomatic or 'subconcussive impacts' of variable magnitudes are common in contact sports. Concerns have risen because athletes returning to activity too soon have an increased risk of prolonged recovery or long-term adverse health consequences. To date, little is understood on a molecular level regarding concussion and subconcussive impacts. Recent research suggests that neuro-inflammatory mechanisms may serve an important role subsequent to concussion and possibly subconcussive impacts. These studies suggest that autoantibodies may be a feasible method of detection for acute concussion and monitoring for changes due to cumulative exposure to subconcussive impacts. Hence, we aimed to profile the IgA autoantibody repertoire in saliva by screening a unique sport-related head trauma biobank. Saliva samples (N=167) were donated by male and female participants enrolled in either the Concussion (24 to 48 hours post-injury) or Subconcussion (non-concussed participants having moderate or high cumulative subconcussive impact exposure) Cohorts. Study design included discovery and verification phases. Discovery aimed to identify new candidate autoimmune targets of IgA. Verification tested whether Concussion and Subconcussion Cohorts increased IgA reactivity and cohorts showed similarities. In each phase, sample testing was randomized. The results show a significant increase in the prevalence of IgA towards protein fragments representing HTR1A, SRRM4 and FAS after concussion and subconcussion. These results may suggest that concussion and subconcussion induce similar physiological effects, especially in terms of immune response. Our study demonstrates that saliva is a potential biofluid for autoantibody detection in concussion and subconcussion. After rigorous confirmation in much larger independent study sets, a validated salivary autoantibody assay could provide a non-subjective quantitative means of assessing concussive and subconcussive events.

Keywords: Other; PROTEOMICS; TRAUMATIC BRAIN INJURY.

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3-27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.

Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. The aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that the knock-down of HDAC2 or the overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.

Keywords: endophilin-B1; hiPSCs; histone deacetylase 2; mitochondria; neuronal differentiation.

Neuroendocrine (NE) differentiation in metastatic castration-resistant prostate cancer (mCRPC) is an increasingly common clinical feature arising from cellular plasticity. We recently characterized two mCRPC phenotypes with NE features: androgen receptor (AR)-positive NE-positive amphicrine prostate cancer (AMPC) and AR-negative small cell or neuroendocrine prostate cancer (SCNPC). Here, we interrogated the regulation of RE1-silencing transcription factor (REST), a transcriptional repressor of neuronal genes, and elucidated molecular programs driving AMPC and SCNPC biology. Analysis of prostate cancer (PC) cell lines, mCRPC specimens, and LuCaP patient-derived xenograft models detected alternative splicing of REST to REST4 and attenuated REST repressor activity in AMPC and SCNPC. The REST locus was also hypermethylated and REST expression was reduced in SCNPC. While serine/arginine repetitive matrix protein 4 (SRRM4) was previously implicated in alternative splicing of REST in mCRPC, we detected SRRM3 expression in REST4-positive, SRRM4-negative AMPC and SCNPC. In CRPC cell lines, SRRM3 induced alternative splicing of REST to REST4 and exacerbated the expression of REST-repressed genes. Furthermore, SRRM3 and SRRM4 expression defined molecular subsets of AMPC and SCNPC across species and tumor types. Two AMPC phenotypes and three SCNPC phenotypes were characterized, denoted either by REST attenuation and ASCL1 activity or by progressive activation of neuronal transcription factor programs, respectively. These results nominate SRRM3 as the principal REST splicing factor expressed in early NE differentiation and provide a framework to molecularly classify diverse NE phenotypes in mCRPC.