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Publication
Journal: Cell
February/22/2015
Abstract
Alternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide "microexons" display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.
Publication
Journal: Molecular Cell
December/21/2011
Abstract
Neurogenesis requires the concerted action of numerous genes that are regulated at multiple levels. However, how different layers of gene regulation are coordinated to promote neurogenesis is not well understood. We show that the neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4) negatively regulates REST (NRSF), a transcriptional repressor of genes required for neurogenesis. nSR100 directly promotes alternative splicing of REST transcripts to produce a REST isoform (REST4) with greatly reduced repressive activity, thereby activating expression of REST targets in neural cells. Conversely, REST directly represses nSR100 in nonneural cells to prevent the activation of neural-specific splicing events. Consistent with a critical role for nSR100 in the inhibition of REST activity, blocking nSR100 expression in the developing mouse brain impairs neurogenesis. Our results thus reveal a fundamental role for direct regulatory interactions between a splicing activator and transcription repressor in the control of the multilayered regulatory programs required for neurogenesis.
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Publication
Journal: European Urology
December/12/2016
Abstract
Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of castration-resistant prostate cancer that typically does not respond to androgen receptor pathway inhibition (ARPI), and its diagnosis is increasing.
To understand how NEPC develops and to identify driver genes to inform therapy for NEPC prevention.
Whole-transcriptome sequencing data were extracted from prostate tumors from two independent cohorts: The Beltran cohort contained 27 adenocarcinoma and five NEPC patient samples, and the Vancouver Prostate Centre cohort contained three patient samples and nine patient-derived xenografts.
A novel bioinformatics tool, comparative alternative splicing detection (COMPAS), was invented to analyze alternative RNA splicing on RNA-sequencing data.
COMPAS identified potential driver genes for NEPC development. Biochemical and biological validations were performed in both prostate cell and tumor models.
More than 66% of the splice events were predicted to be regulated by the RNA splicing factor serine/arginine repetitive matrix 4 (SRRM4). In vitro and in vivo evidence confirmed that one SRRM4 target gene was the RE1 silencing transcription factor (REST), a master regulator of neurogenesis. Moreover, SRRM4 strongly stimulated adenocarcinoma cells to express NEPC biomarkers, and this effect was exacerbated by ARPI. ARPI combined with a gain of SRRM4-induced adenocarcinoma cells to assume multicellular spheroid morphology and was essential in establishing progressive NEPC xenografts. These SRRM4 actions were further enhanced by loss of function of TP53.
SRRM4 drives NEPC progression. This knowledge may guide the development of novel therapeutics aimed at NEPC.
Using next-generation RNA sequencing and our newly developed bioinformatics tool, we identified a neuroendocrine prostate cancer (NEPC)-specific RNA splicing signature that is predominantly controlled by serine/arginine repetitive matrix 4 (SRRM4). We confirmed that SRRM4 drives NEPC progression, and we propose SRRM4 as a potential therapeutic target for NEPC.
Publication
Journal: Clinical Cancer Research
July/31/2016
Abstract
OBJECTIVE
The neuroendocrine phenotype is associated with the development of metastatic castration-resistant prostate cancer (CRPC). Our objective was to characterize the molecular features of the neuroendocrine phenotype in CRPC.
METHODS
Expression of chromogranin A (CHGA), synaptophysin (SYP), androgen receptor (AR), and prostate-specific antigen (PSA) was analyzed by IHC in 155 CRPC metastases from 50 patients and in 24 LuCaP prostate cancer patient-derived xenografts (PDX). Seventy-one of 155 metastases and the 24 LuCaP xenograft lines were analyzed by whole-genome microarrays. REST splicing was verified by PCR.
RESULTS
Coexpression of CHGA and SYP in >30% of cells was observed in 22 of 155 metastases (9 patients); 11 of the 22 metastases were AR(+)/PSA(+) (6 patients), 11/22 were AR-/PSA- (4 patients), and 4/24 LuCaP PDXs were AR(-)/PSA(-). By IHC, of the 71 metastases analyzed by whole-genome microarrays, 5 metastases were CHGA(+)/SYP(+)/AR(-), and 5 were CHGA(+)/SYP(+)/AR(+). Only CHGA(+)/SYP(+) metastases had a neuroendocrine transcript signature. The neuronal transcriptional regulator SRRM4 transcript was associated with the neuroendocrine signature in CHGA(+)/SYP(+) metastases and all CHGA(+)/SYP(+) LuCaP xenografts. In addition, expression of SRRM4 in LuCaP neuroendocrine xenografts correlated with a splice variant of REST that lacks the transcriptional repressor domain.
CONCLUSIONS
(i) Metastatic neuroendocrine status can be heterogeneous in the same patient, (ii) the CRPC neuroendocrine molecular phenotype can be defined by CHGA(+)/SYP(+) dual positivity, (iii) the neuroendocrine phenotype is not necessarily associated with the loss of AR activity, and (iv) the splicing of REST by SRRM4 could promote the neuroendocrine phenotype in CRPC.
Publication
Journal: Molecular Cell
December/28/2014
Abstract
The vertebrate and neural-specific Ser/Arg (SR)-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells while weakening 3' splice site recognition and contributing to exon skipping in nonneural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis.
Publication
Journal: PLoS Genetics
March/21/2013
Abstract
Sensory hair cells are essential for hearing and balance. Their development from epithelial precursors has been extensively characterized with respect to transcriptional regulation, but not in terms of posttranscriptional influences. Here we report on the identification and functional characterization of an alternative-splicing regulator whose inactivation is responsible for defective hair-cell development, deafness, and impaired balance in the spontaneous mutant Bronx waltzer (bv) mouse. We used positional cloning and transgenic rescue to locate the bv mutation to the splicing factor-encoding gene Ser/Arg repetitive matrix 4 (Srrm4). Transcriptome-wide analysis of pre-mRNA splicing in the sensory patches of embryonic inner ears revealed that specific alternative exons were skipped at abnormally high rates in the bv mice. Minigene experiments in a heterologous expression system confirmed that these skipped exons require Srrm4 for inclusion into the mature mRNA. Sequence analysis and mutagenesis experiments showed that the affected transcripts share a novel motif that is necessary for the Srrm4-dependent alternative splicing. Functional annotations and protein-protein interaction data indicated that the encoded proteins cluster in the secretion and neurotransmission pathways. In addition, the splicing of a few transcriptional regulators was found to be Srrm4 dependent, and several of the genes known to be targeted by these regulators were expressed at reduced levels in the bv mice. Although Srrm4 expression was detected in neural tissues as well as hair cells, analyses of the bv mouse cerebellum and neocortex failed to detect splicing defects. Our data suggest that Srrm4 function is critical in the hearing and balance organs, but not in all neural tissues. Srrm4 is the first alternative-splicing regulator to be associated with hearing, and the analysis of bv mice provides exon-level insights into hair-cell development.
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Publication
Journal: Genes and Development
May/28/2015
Abstract
Alternative splicing (AS) generates vast transcriptomic complexity in the vertebrate nervous system. However, the extent to which trans-acting splicing regulators and their target AS regulatory networks contribute to nervous system development is not well understood. To address these questions, we generated mice lacking the vertebrate- and neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4). Loss of nSR100 impairs development of the central and peripheral nervous systems in part by disrupting neurite outgrowth, cortical layering in the forebrain, and axon guidance in the corpus callosum. Accompanying these developmental defects are widespread changes in AS that primarily result in shifts to nonneural patterns for different classes of splicing events. The main component of the altered AS program comprises 3- to 27-nucleotide (nt) neural microexons, an emerging class of highly conserved AS events associated with the regulation of protein interaction networks in developing neurons and neurological disorders. Remarkably, inclusion of a 6-nt, nSR100-activated microexon in Unc13b transcripts is sufficient to rescue a neuritogenesis defect in nSR100 mutant primary neurons. These results thus reveal critical in vivo neurodevelopmental functions of nSR100 and further link these functions to a conserved program of neuronal microexon splicing.
Publication
Journal: Cerebral Cortex
May/5/2016
Abstract
Alternative splicing in the brain is dynamic and instrumental to adaptive changes in response to stimuli. Lysine-specific demethylase 1 (LSD1/KDM1A) is a ubiquitously expressed histone H3Lys4 demethylase that acts as a transcriptional co-repressor in complex with its molecular partners CoREST and HDAC1/2. In mammalian brain, alternative splicing of LSD1 mini-exon E8a gives rise to neuroLSD1, a neurospecific isoform that, upon phosphorylation, acts as a dominant-negative causing disassembly of the co-repressor complex and de-repression of target genes. Here we show that the LSD1/neuroLSD1 ratio changes in response to neuronal activation and such effect is mediated by neurospecific splicing factors NOVA1 and nSR100/SRRM4 together with a novel cis-silencer. Indeed, we found that, in response to epileptogenic stimuli, downregulation of NOVA1 reduces exon E8a splicing and expression of neuroLSD1. Using behavioral and EEG analyses we observed that neuroLSD1-specific null mice are hypoexcitable and display decreased seizure susceptibility. Conversely, in a mouse model of Rett syndrome characterized by hyperexcitability, we measured higher levels of NOVA1 protein and upregulation of neuroLSD1. In conclusion, we propose that, in the brain, correct ratio between LSD1 and neuroLSD1 contributes to excitability and, when altered, could represent a pathogenic event associated with neurological disorders involving altered E/I.
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Publication
Journal: Molecular Cell
September/4/2017
Abstract
A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.
Publication
Journal: Human Genetics
November/16/2017
Abstract
Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.
Publication
Journal: Molecular Cancer Research
May/27/2014
Abstract
Small cell lung cancer (SCLC) is a highly malignant form of cancer, which originates from primitive neuroendocrine cells in the lung. SCLC cells express several autocrine neurotransmitters/neuropeptides and their respective receptors. Expression of these neuronal markers is frequently regulated by RE1-silencing transcription factor (REST). In SCLC cells, an SCLC-specific isoform of REST (sREST) is highly expressed, whereas REST expression is undetectable, suggesting that the expression of sREST correlates with the pathogenesis of SCLC. Expression of sREST, which is derived through alternative splicing of REST, is abnormally regulated in SCLC cells, but the mechanism is unknown. Most recently, nSR100 (SRRM4) was described as an activator of REST alternative splicing. We now show that nSR100 is highly expressed in SCLC cells correlating with high sREST and low REST expression. Adhesion to the extracellular matrix (ECM) is thought to enhance tumorigenicity and confer resistance to apoptosis. Interestingly, nSR100 expression is enhanced in cells grown with ECM. Overexpression of REST caused repression of sREST and nSR100, the latter containing RE1 element controlled by REST. Culturing the SCLC cell line NCI-N417 cells with ECM also upregulated RE1-containing gene, the voltage-gated calcium channel subunit. Inhibition of the PI3K/Akt/mTOR pathway by LY294002 induced nSR100 expression, whereas the specific MEK/ERK inhibitor U0126 inhibited nSR100 expression. Repressing nSR100 by siRNA effectively repressed sREST, and conversely increased REST in NCI-N417 cells. Taken together, this report clarifies the ECM-dependent signaling pathway that impacts nSR100 expression and its regulation of alternative splicing in SCLC.
CONCLUSIONS
The splicing factor nSR100 may be novel SCLC-specific biomarker, as well as a therapeutic target.
Publication
Journal: Alzheimer's and Dementia
November/12/2018
Abstract
BACKGROUND
Genetic associations for endophenotypes of Alzheimer's disease (AD) in cognitive stages preceding AD have not been thoroughly evaluated.
METHODS
We conducted genome-wide association studies for AD-related endophenotypes including hippocampal volume, logical memory scores, and cerebrospinal fluid Aβ42 and total/phosphorylated tau in cognitively normal (CN), mild cognitive impairment, and AD dementia subjects from the Alzheimer's Disease Neuroimaging Initiative study.
RESULTS
In CN subjects, study-wide significant (P < 8.3 × 10-9) loci were identified for total tau near SRRM4 and C14orf79 and for hippocampal volume near MTUS1. In mild cognitive impairment subjects, study-wide significant association was found with single nucleotide polymorphisms (SNPs) near ZNF804B for logical memory test of delayed recall scores. We found consistent expression patterns of C14orf40 and MTUS1 in carriers with risk alleles of expression SNPs and in brains of AD patients, compared with in the noncarriers and in brains of controls.
CONCLUSIONS
Our findings for AD-related brain changes before AD provide insight about early AD-related biological processes.
Publication
Journal: Oncotarget
May/24/2017
Abstract
Although potent androgen receptor pathway inhibitors (ARPI) improve overall survival of metastatic prostate cancer patients, treatment-induced neuroendocrine prostate cancer (t-NEPC) as a consequence of the selection pressures of ARPI is becoming a more common clinical issue. Improved understanding of the molecular biology of t-NEPC is essential for the development of new effective management approaches for t-NEPC. In this study, we identify a splice variant of the MYST/Esa1-associated factor 6 (MEAF6) gene, MEAF6-1, that is highly expressed in both t-NEPC tumor biopsies and neuroendocrine cell lines of prostate and lung cancers. We show that MEAF6-1 splicing is stimulated by neuronal RNA splicing factor SRRM4. Rather than inducing neuroendocrine trans-differentiation of cells in prostate adenocarcinoma, MEAF6-1 upregulation stimulates cell proliferation, anchorage-independent cell growth, invasion and xenograft tumor growth. Gene microarray identifies that these MEAF6-1 actions are in part mediated by the ID1 and ID3 genes. These findings suggest that the MEAF6-1 variant does not induce neuroendocrine differentiation of prostate cancer cells, but rather facilitates t-NEPC progression by increasing the proliferation rate of cells that have acquired neuroendocrine phenotypes.
Publication
Journal: Prostate
December/9/2018
Abstract
Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of castrate-resistant prostate cancer characterized by poor patient outcome. Whole transcriptome sequencing analyses identified a NEPC-specific RNA splicing program that is predominantly controlled by the SRRM4 gene, suggesting that SRRM4 drives NEPC development. However, whether SRRM4 expression in patients may aid pathologists in diagnosing NEPC and predicting patient survival remains to be determined. In this study, we have applied RNA in situ hybridization and immunohistochemistry assays to measure the expressions of SRRM4, NEPC markers (SYP, CD56, and CHGA), and adenocarcinoma (AdPC) markers (AR, PSA) in a series of tissue microarrays constructed from castrate-resistant prostate tumors, treatment-naïve tumors collected from radical prostatectomy, and tumors treated with neoadjuvant hormonal therapy (NHT) for 0-12 months. Three pathologists also independently evaluated tumor histology and NEPC marker status. Here, we report that SRRM4 in castrate-resistant tumors is highly expressed in NEPC, strongly correlated with SYP, CD56, and CHGA expressions (Pearson correlation r = 0.883, 0.675, and 0.881; P < 0.0001) and negatively correlated with AR and PSA expressions (Pearson correlation r = -0.544 and -0.310; P < 0.05). Overall survival is 12.3 months for patients with SRRM4 positive tumors, comparing to 23 months for patients with SRRM4 negative tumors. In treatment-naïve AdPC, low SRRM4 expression is detected in ∼16% tumor cores. It correlates with SYP and CHGA expressions, but not Gleason scores. AdPC treated with >7 month NHT has significantly higher SRRM4 expression. Based on these findings, we conclude that SRRM4 expression in castrate-resistant tumors is highly correlated with NEPC and poor patient survival. It may serve as a diagnosis and prognosis biomarker of NEPC.
Publication
Journal: Molecular Cell
November/1/2018
Abstract
Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.
Publication
Journal: Nature Ecology and Evolution
March/5/2019
Abstract
The mechanisms by which entire programmes of gene regulation emerged during evolution are poorly understood. Neuronal microexons represent the most conserved class of alternative splicing in vertebrates, and are critical for proper brain development and function. Here, we discover neural microexon programmes in non-vertebrate species and trace their origin to bilaterian ancestors through the emergence of a previously uncharacterized 'enhancer of microexons' (eMIC) protein domain. The eMIC domain originated as an alternative, neural-enriched splice isoform of the pan-eukaryotic Srrm2/SRm300 splicing factor gene, and subsequently became fixed in the vertebrate and neuronal-specific splicing regulator Srrm4/nSR100 and its paralogue Srrm3. Remarkably, the eMIC domain is necessary and sufficient for microexon splicing, and functions by interacting with the earliest components required for exon recognition. The emergence of a novel domain with restricted expression in the nervous system thus resulted in the evolution of splicing programmes that qualitatively expanded the neuronal molecular complexity in bilaterians.
Publication
Journal: Translational Psychiatry
August/18/2020
Abstract
Autism spectrum disorder (ASD) has phenotypically and genetically heterogeneous characteristics. A simulation study demonstrated that attempts to categorize patients with a complex disease into more homogeneous subgroups could have more power to elucidate hidden heritability. We conducted cluster analyses using the k-means algorithm with a cluster number of 15 based on phenotypic variables from the Simons Simplex Collection (SSC). As a preliminary study, we conducted a conventional genome-wide association study (GWAS) with a data set of 597 ASD cases and 370 controls. In the second step, we divided cases based on the clustering results and conducted GWAS in each of the subgroups vs controls (cluster-based GWAS). We also conducted cluster-based GWAS on another SSC data set of 712 probands and 354 controls in the replication stage. In the preliminary study, which was conducted in conventional GWAS design, we observed no significant associations. In the second step of cluster-based GWASs, we identified 65 chromosomal loci, which included 30 intragenic loci located in 21 genes and 35 intergenic loci that satisfied the threshold of P < 5.0 × 10-8. Some of these loci were located within or near previously reported candidate genes for ASD: CDH5, CNTN5, CNTNAP5, DNAH17, DPP10, DSCAM, FOXK1, GABBR2, GRIN2A5, ITPR1, NTM, SDK1, SNCA, and SRRM4. Of these 65 significant chromosomal loci, rs11064685 located within the SRRM4 gene had a significantly different distribution in the cases vs controls in the replication cohort. These findings suggest that clustering may successfully identify subgroups with relatively homogeneous disease etiologies. Further cluster validation and replication studies are warranted in larger cohorts.
Publication
Journal: Scientific Reports
February/19/2017
Abstract
Alternative splicing gives rise to diversity of the proteome, and it is especially prevalent in the mammalian nervous system. Indeed, many factors that control the splicing process govern nervous system development. Among such factors, SRRM4 is an important regulator of aspects of neural differentiation including neurite outgrowth. The mechanism by which SRRM4 regulates neurite outgrowth has remained poorly understood, however. We now show that SRRM4 regulates the splicing of protrudin gene (Zfyve27) transcripts in neuronal cells. SRRM4 was found to promote splicing of protrudin pre-mRNA so as to include a microexon (exon L) encoding seven amino acids in a neuron-specific manner. The resulting protein (protrudin-L) promotes neurite outgrowth during neurogenesis. Depletion of SRRM4 in Neuro2A cells impaired inclusion of exon L in protrudin mRNA, resulting in the generation of a shorter protein isoform (protrudin-S) that is less effective at promoting neurite extension. SRRM4 was found to recognize a UGC motif that is located immediately upstream of exon L and is necessary for inclusion of exon L in the mature transcript. Deletion of exon L in Neuro2A or embryonic stem cells inhibited neurite outgrowth. Our results suggest that SRRM4 controls neurite outgrowth through regulation of alternative splicing of protrudin transcripts.
Publication
Journal: Neoplasia
May/13/2020
Abstract
Neuroendocrine prostate cancer (NEPC) is the most virulent form of prostate cancer. Importantly, our recent work examining metastatic biopsy samples demonstrates NEPC is increasing in frequency. In contrast to prostate adenocarcinomas that express a luminal gene expression program, NEPC tumors express a neuronal gene expression program. Despite this distinction, the diagnosis of NEPC is often challenging, demonstrating an urgent need to identify new biomarkers and therapeutic targets. Our prior work demonstrated that the histone demethylase LSD1 (KDM1A) is important for survival of prostate adenocarcinomas, but little was known about LSD1's role in NEPC. Recently, a neural-specific transcript variant of LSD1-LSD1+8a-was discovered and demonstrated to activate neuronal gene expression in neural cells. The splicing factor SRRM4 was previously shown to promote LSD1+8a splicing in neuronal cells, and SRRM4 promotes NEPC differentiation and cell survival. Therefore, we sought to determine if LSD1+8a might play a role in NEPC and whether LSD1+8a splicing was linked to SRRM4. To investigate a potential role for LSD1+8a in NEPC, we examined a panel of prostate adenocarcinoma and NEPC patient-derived xenografts and metastatic biopsies. LSD1+8a was expressed exclusively in NEPC samples and correlated significantly with elevated expression of SRRM4. Using SRRM4-overexpressing cell lines, we determined that SRRM4 mediates alternative splicing of LSD1+8a. Finally, using gain of function studies, we confirmed that LSD1+8a and SRRM4 co-regulate target genes distinct from canonical LSD1. Our findings suggest further study of the interplay between SRRM4 and LSD1+8a and mechanisms by which LSD1+8a regulates gene expression in NEPC is warranted.
Publication
Journal: Developmental Biology
August/3/2015
Abstract
The vertebrate brain arises from the complex organization of millions of neurons. Neurogenesis encompasses not only cell fate specification from neural stem cells, but also the terminal molecular and morphological maturation of neurons at correct positions within the brain. RE1-silencing transcription factor (Rest) is expressed in non-neural tissues and neuronal progenitors where it inhibits the terminal maturation of neurons by repressing hundreds of neuron-specific genes. Here we show that Rest repression of maturation is intimately linked with the migratory capability of zebrafish facial branchiomotor neurons (FBMNs), which undergo a characteristic tangential migration from hindbrain rhombomere (r) 4 to r6/r7 during development. We establish that FBMN migration is increasingly disrupted as Rest is depleted in zebrafish rest mutant embryos, such that around two-thirds of FBMNs fail to complete migration in mutants depleted of both maternal and zygotic Rest. Although Rest is broadly expressed, we show that de-repression or activation of Rest target genes only within FBMNs is sufficient to disrupt their migration. We demonstrate that this migration defect is due to precocious maturation of FBMNs, based on both morphological and molecular criteria. We further show that the Rest target gene and alternative splicing factor srrm4 is a key downstream regulator of maturation; Srrm4 knockdown partially restores the ability of FBMNs to migrate in rest mutants while preventing their precocious morphological maturation. Rest must localize to the nucleus to repress its targets, and its subcellular localization is highly regulated: we show that targeting Rest specifically to FBMN nuclei rescues FBMN migration in Rest-deficient embryos. We conclude that Rest functions in FBMN nuclei to inhibit maturation until the neurons complete their migration.
Publication
Journal: Cell Reports
April/17/2019
Abstract
A truncating mutation in the mouse Srrm4 gene, which encodes a neuronal splicing factor, causes alternative splicing defects selectively in the ear. The mechanism by which splicing is preserved in the brain of these mice is not known. Here, we show that SRRM3 limits the Srrm4 mutation-associated defects to the ear and that, in cortical neurons, overlapping SRRM3-SRRM4 activity regulates the development of interneuronal inhibition. In vitro, SRRM3 and SRRM4 regulate the same splicing events, but a mutation in mouse Srrm3 causes tremors and mild defects in neuronal alternative splicing, demonstrating unique SRRM3 roles in vivo. Mice harboring mutations in both Srrm3 and Srrm4 die neonatally and exhibit severe splicing defects. In these mice, splicing alterations prevent inactivation of the gene repressor REST, which maintains immature excitatory GABAergic neurotransmission by repressing K-Cl cotransporter 2. Thus, our data reveal that SRRM3 and SRRM4 act redundantly to regulate GABAergic neurotransmission by inactivating REST.
Publication
Journal: EBioMedicine
November/13/2018
Abstract
BACKGROUND
Prostate adenocarcinoma (AdPC) cells can undergo lineage switching to neuroendocrine cells and develop into therapy-resistant neuroendocrine prostate cancer (NEPC). While genomic/epigenetic alterations are shown to induce neuroendocrine differentiation via an intermediate stem-like state, RNA splicing factor SRRM4 can transform AdPC cells into NEPC xenografts through a direct neuroendocrine transdifferentiation mechanism. Whether SRRM4 can also regulate a stem-cell gene network for NEPC development remains unclear.
METHODS
Multiple AdPC cell models were transduced by lentiviral vectors encoding SRRM4. SRRM4-mediated RNA splicing and neuroendocrine differentiation of cells and xenografts were determined by qPCR, immunoblotting, and immunohistochemistry. Cell morphology, proliferation, and colony formation rates were also studied. SRRM4 transcriptome in the DU145 cell model was profiled by AmpliSeq and analyzed by gene enrichment studies.
RESULTS
SRRM4 induces an overall NEPC-specific RNA splicing program in multiple cell models but creates heterogeneous transcriptomes. SRRM4-transduced DU145 cells present the most dramatic neuronal morphological changes, accelerated cell proliferation, and enhanced resistance to apoptosis. The derived xenografts show classic phenotypes similar to clinical NEPC. Whole transcriptome analyses further reveal that SRRM4 induces a pluripotency gene network consisting of the stem-cell differentiation gene, SOX2. While SRRM4 overexpression enhances SOX2 expression in both time- and dose-dependent manners in DU145 cells, RNA depletion of SOX2 compromises SRRM4-mediated stimulation of pluripotency genes. More importantly, this SRRM4-SOX2 axis is present in a subset of NEPC patient cohorts, patient-derived xenografts, and clinically relevant transgenic mouse models.
CONCLUSIONS
We report a novel mechanism by which SRRM4 drives NEPC progression via a pluripotency gene network. FUND: Canadian Institutes of Health Research, National Nature Science Foundation of China, and China Scholar Council.
Publication
Journal: EBioMedicine
September/23/2018
Abstract
Treatment-induced neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer (PCa) that becomes more prevalent when hormonal therapy, chemotherapy, or radiation therapy is applied to patients with metastatic prostate adenocarcinoma (AdPC). How AdPC cells survive these anti-cancer therapies and progress into t-NEPC remains unclear. By comparing the whole transcriptomes between AdPC and t-NEPC, we identified Bif-1, an apoptosis-associated gene, which undergoes alternative RNA splicing in t-NEPC. We found that while Bif-1a is the predominant variant of the Bif-1 gene in AdPC, two neural-specific variants, Bif-1b and Bif-1c, are highly expressed in t-NEPC patients, patient derived xenografts, and cell models. The neural-specific RNA splicing factor, SRRM4, promotes Bif-1b and Bif-1c splicing, and the expression of SRRM4 in tumors is strongly associated with Bif-1b/-1c levels. Furthermore, we showed that Bif-1a is pro-apoptotic, while Bif-1b and Bif-1c are anti-apoptotic in PCa cells under camptothecin and UV light irritation treatments. Taken together, our data indicate that SRRM4 regulates alternative RNA splicing of the Bif-1 gene that enables PCa cells resistant to apoptotic stimuli under anti-cancer therapies, and may contribute to AdPC progression into t-NEPC.
Publication
Journal: RNA Biology
September/27/2019
Abstract
Neuronal microexons represent the most highly conserved class of alternative splicing events and their timed expression shapes neuronal biology, including neuronal commitment and differentiation. The six-nt microexon 34' is included in the neuronal form of TAF1 mRNA, which encodes the largest subunit of the basal transcription factor TFIID. In this study, we investigate the tissue distribution of TAF1-34' mRNA and protein and the mechanism responsible for its neuronal-specific splicing. Using isoform-specific RNA probes and antibodies, we observe that canonical TAF1 and TAF1-34' have different distributions in the brain, which distinguish proliferating from post-mitotic neurons. Knockdown and ectopic expression experiments demonstrate that the neuronal-specific splicing factor SRRM4/nSR100 promotes the inclusion of microexon 34' into TAF1 mRNA, through the recognition of UGC sequences in the poly-pyrimidine tract upstream of the regulated microexon. These results show that SRRM4 regulates temporal and spatial expression of alternative TAF1 mRNAs to generate a neuronal-specific TFIID complex.
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