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1.
PLoS Genet ; 18(9): e1010416, 2022 09.
Article in English | MEDLINE | ID: mdl-36129965

ABSTRACT

Control over gene expression is exerted, in multiple stages of spermatogenesis, at the post-transcriptional level by RNA binding proteins (RBPs). We identify here an essential role in mammalian spermatogenesis and male fertility for 'RNA binding protein 46' (RBM46). A highly evolutionarily conserved gene, Rbm46 is also essential for fertility in both flies and fish. We found Rbm46 expression was restricted to the mouse germline, detectable in males in the cytoplasm of premeiotic spermatogonia and meiotic spermatocytes. To define its requirement for spermatogenesis, we generated Rbm46 knockout (KO, Rbm46-/-) mice; although male Rbm46-/- mice were viable and appeared grossly normal, they were infertile. Testes from adult Rbm46-/- mice were small, with seminiferous tubules containing only Sertoli cells and few undifferentiated spermatogonia. Using genome-wide unbiased high throughput assays RNA-seq and 'enhanced crosslinking immunoprecipitation' coupled with RNA-seq (eCLIP-seq), we discovered RBM46 could bind, via a U-rich conserved consensus sequence, to a cohort of mRNAs encoding proteins required for completion of differentiation and subsequent meiotic initiation. In summary, our studies support an essential role for RBM46 in regulating target mRNAs during spermatogonia differentiation prior to the commitment to meiosis in mice.


Subject(s)
RNA-Binding Proteins/metabolism , Spermatogenesis , Spermatogonia , Animals , Cell Differentiation/genetics , Male , Mammals/genetics , Meiosis/genetics , Mice , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Spermatocytes/metabolism , Spermatogenesis/genetics , Spermatogonia/metabolism , Testis
2.
Development ; 147(21)2020 04 30.
Article in English | MEDLINE | ID: mdl-32253237

ABSTRACT

Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor Esrp1 have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new Esrp1 mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in Esrp1-/- embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using Esrp1 mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients.


Subject(s)
Cleft Lip/pathology , Cleft Palate/pathology , Epithelium/pathology , Mesoderm/pathology , RNA-Binding Proteins/metabolism , Signal Transduction , Alternative Splicing/genetics , Animals , Cell Proliferation , Cleft Lip/embryology , Cleft Lip/genetics , Cleft Palate/embryology , Cleft Palate/genetics , Ectoderm/embryology , Ectoderm/metabolism , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Epithelium/embryology , Face , Gene Expression Regulation, Developmental , Genes, Reporter , Mesoderm/embryology , Mice, Knockout , Organogenesis/genetics , Palate/embryology , Palate/pathology
3.
Development ; 147(24)2020 12 23.
Article in English | MEDLINE | ID: mdl-33234718

ABSTRACT

Irf6 and Esrp1 are important for palate development across vertebrates. In zebrafish, we found that irf6 regulates the expression of esrp1 We detailed overlapping Irf6 and Esrp1/2 expression in mouse orofacial epithelium. In zebrafish, irf6 and esrp1/2 share expression in periderm, frontonasal ectoderm and oral epithelium. Genetic disruption of irf6 and esrp1/2 in zebrafish resulted in cleft of the anterior neurocranium. The esrp1/2 mutant also developed cleft of the mouth opening. Lineage tracing of cranial neural crest cells revealed that the cleft resulted not from migration defect, but from impaired chondrogenesis. Analysis of aberrant cells within the cleft revealed expression of sox10, col1a1 and irf6, and these cells were adjacent to krt4+ and krt5+ cells. Breeding of mouse Irf6; Esrp1; Esrp2 compound mutants suggested genetic interaction, as the triple homozygote and the Irf6; Esrp1 double homozygote were not observed. Further, Irf6 heterozygosity reduced Esrp1/2 cleft severity. These studies highlight the complementary analysis of Irf6 and Esrp1/2 in mouse and zebrafish, and identify a unique aberrant cell population in zebrafish expressing sox10, col1a1 and irf6 Future work characterizing this cell population will yield additional insight into cleft pathogenesis.


Subject(s)
Interferon Regulatory Factors/genetics , Maxillofacial Development/genetics , Morphogenesis/genetics , RNA-Binding Proteins/genetics , Animals , Ectoderm/growth & development , Ectoderm/metabolism , Epithelium/growth & development , Gene Expression Regulation, Developmental/genetics , Humans , Mice , Mutation/genetics , Neural Crest/growth & development , SOXE Transcription Factors/genetics , Zebrafish , Zebrafish Proteins/genetics
4.
Nat Methods ; 16(4): 307-310, 2019 04.
Article in English | MEDLINE | ID: mdl-30923373

ABSTRACT

A major limitation of RNA sequencing (RNA-seq) analysis of alternative splicing is its reliance on high sequencing coverage. We report DARTS (https://github.com/Xinglab/DARTS), a computational framework that integrates deep-learning-based predictions with empirical RNA-seq evidence to infer differential alternative splicing between biological samples. DARTS leverages public RNA-seq big data to provide a knowledge base of splicing regulation via deep learning, thereby helping researchers better characterize alternative splicing using RNA-seq datasets even with modest coverage.


Subject(s)
Deep Learning , RNA Splicing , RNA/analysis , Sequence Analysis, RNA , Algorithms , Alternative Splicing , Bayes Theorem , Epigenomics , Exons , Gene Expression Profiling , Gene Expression Regulation , Hep G2 Cells , High-Throughput Nucleotide Sequencing , Humans , K562 Cells , Models, Statistical , RNA/genetics , Reproducibility of Results , Signal Processing, Computer-Assisted
5.
Mol Cell ; 54(6): 903-904, 2014 Jun 19.
Article in English | MEDLINE | ID: mdl-24950376

ABSTRACT

Using Caenorhabditis elegans as a model system, Norris et al. (2014) define complex combinatorial regulation of alternative splicing at single-neuron resolution and illustrate functional coherence among components of a splicing regulatory network controlled by a neuronal splicing factor.


Subject(s)
Alternative Splicing/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/genetics , Cholinergic Neurons/cytology , GABAergic Neurons/cytology , RNA-Binding Proteins/physiology , Synaptic Transmission/genetics , Syntaxin 1/genetics , Animals
6.
Am J Hum Genet ; 102(6): 1143-1157, 2018 06 07.
Article in English | MEDLINE | ID: mdl-29805042

ABSTRACT

Non-syndromic cleft lip with or without cleft palate (NS-CL/P) is one of the most common human birth defects and is generally considered a complex trait. Despite numerous loci identified by genome-wide association studies, the effect sizes of common variants are relatively small, with much of the presumed genetic contribution remaining elusive. We report exome-sequencing results in 209 people from 72 multi-affected families with pedigree structures consistent with autosomal-dominant inheritance and variable penetrance. Herein, pathogenic variants are described in four genes encoding components of the p120-catenin complex (CTNND1, PLEKHA7, PLEKHA5) and an epithelial splicing regulator (ESRP2), in addition to the known CL/P-associated gene, CDH1, which encodes E-cadherin. The findings were also validated in a second cohort of 497 people with NS-CL/P, comprising small families and singletons with pathogenic variants in these genes identified in 14% of multi-affected families and 2% of the replication cohort of smaller families. Enriched expression of each gene/protein in human and mouse embryonic oro-palatal epithelia, demonstration of functional impact of CTNND1 and ESRP2 variants, and recapitulation of the CL/P spectrum in Ctnnd1 knockout mice support a causative role in CL/P pathogenesis. These data show that primary defects in regulators of epithelial cell adhesion are the most significant contributors to NS-CL/P identified to date and that inherited and de novo single gene variants explain a substantial proportion of NS-CL/P.


Subject(s)
Cadherins/genetics , Catenins/genetics , Cleft Lip/genetics , Cleft Palate/genetics , Genetic Predisposition to Disease , Mutation/genetics , Alleles , Amino Acid Sequence , Animals , Biotinylation , Epithelium/metabolism , Epithelium/pathology , Female , Gene Deletion , Humans , Infant , Infant, Newborn , Male , Mice , Palate/pathology , Pedigree , Syndrome , Exome Sequencing , Delta Catenin
7.
Nature ; 499(7457): 172-7, 2013 Jul 11.
Article in English | MEDLINE | ID: mdl-23846655

ABSTRACT

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.


Subject(s)
Gene Expression Regulation/genetics , Nucleotide Motifs/genetics , RNA-Binding Proteins/metabolism , Autistic Disorder/genetics , Base Sequence , Binding Sites/genetics , Conserved Sequence/genetics , Eukaryotic Cells/metabolism , Humans , Molecular Sequence Data , Protein Structure, Tertiary/genetics , RNA Splicing Factors , RNA Stability/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics
8.
Biochim Biophys Acta Mol Basis Dis ; 1864(4 Pt A): 1060-1071, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29309924

ABSTRACT

Metastatic breast cancer is a leading cause of cancer-related deaths in women worldwide. Patients with triple negative breast cancer (TNBCs), a highly aggressive tumor subtype, have a particularly poor prognosis. Multiple reports demonstrate that altered content of the multicopy mitochondrial genome (mtDNA) in primary breast tumors correlates with poor prognosis. We earlier reported that mtDNA copy number reduction in breast cancer cell lines induces an epithelial-mesenchymal transition associated with metastasis. However, it is unknown whether the breast tumor subtypes (TNBC, Luminal and HER2+) differ in the nature and amount of mitochondrial defects and if mitochondrial defects can be used as a marker to identify tumors at risk for metastasis. By analyzing human primary tumors, cell lines and the TCGA dataset, we demonstrate a high degree of variability in mitochondrial defects among the tumor subtypes and TNBCs, in particular, exhibit higher frequency of mitochondrial defects, including reduced mtDNA content, mtDNA sequence imbalance (mtRNR1:ND4), impaired mitochondrial respiration and metabolic switch to glycolysis which is associated with tumorigenicity. We identified that genes involved in maintenance of mitochondrial structural and functional integrity are differentially expressed in TNBCs compared to non-TNBC tumors. Furthermore, we identified a subset of TNBC tumors that contain lower expression of epithelial splicing regulatory protein (ESRP)-1, typical of metastasizing cells. The overall impact of our findings reported here is that mitochondrial heterogeneity among TNBCs can be used to identify TNBC patients at risk of metastasis and the altered metabolism and metabolic genes can be targeted to improve chemotherapeutic response.


Subject(s)
DNA, Mitochondrial , Mitochondria , Mitochondrial Proteins , Neoplasm Proteins , RNA-Binding Proteins , Triple Negative Breast Neoplasms , Cell Line, Tumor , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Female , Humans , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology
9.
Mol Cell ; 33(5): 591-601, 2009 Mar 13.
Article in English | MEDLINE | ID: mdl-19285943

ABSTRACT

Cell-type-specific expression of epithelial and mesenchymal isoforms of Fibroblast Growth Factor Receptor 2 (FGFR2) is achieved through tight regulation of mutually exclusive exons IIIb and IIIc, respectively. Using an application of cell-based cDNA expression screening, we identified two paralogous epithelial cell-type-specific RNA-binding proteins that are essential regulators of FGFR2 splicing. Ectopic expression of either protein in cells that express FGFR2-IIIc caused a switch in endogenous FGFR2 splicing to the epithelial isoform. Conversely, knockdown of both factors in cells that express FGFR2-IIIb by RNA interference caused a switch from the epithelial to mesenchymal isoform. These factors also regulate splicing of CD44, p120-Catenin (CTNND1), and hMena (ENAH), three transcripts that undergo changes in splicing during the epithelial-to-mesenchymal transition (EMT). These studies suggest that Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are coordinators of an epithelial cell-type-specific splicing program.


Subject(s)
Alternative Splicing , Epithelial Cells/metabolism , RNA-Binding Proteins/metabolism , Receptor, Fibroblast Growth Factor, Type 2/genetics , Amino Acid Sequence , Animals , Base Sequence , Catenins , Cell Adhesion Molecules/metabolism , Cell Line , Exons , Gene Expression Regulation , Humans , Hyaluronan Receptors/metabolism , Introns , Mice , Mice, Inbred C57BL , Microfilament Proteins/metabolism , Molecular Sequence Data , Phosphoproteins/metabolism , Protein Isoforms , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Transduction, Genetic , Delta Catenin
10.
Dev Dyn ; 245(10): 991-1000, 2016 10.
Article in English | MEDLINE | ID: mdl-27404344

ABSTRACT

BACKGROUND: Abnormalities in ureteric bud (UB) branching morphogenesis lead to congenital anomalies of the kidney and reduced nephron numbers associated with chronic kidney disease (CKD) and hypertension. Previous studies showed that the epithelial fibroblast growth factor receptor 2 (Fgfr2) IIIb splice variant supports ureteric morphogenesis in response to ligands from the metanephric mesenchyme during renal organogenesis. The epithelial-specific splicing regulator Esrp1 is required for expression of Fgfr2-IIIb and other epithelial-specific splice variants. Our objective was to determine whether Esrp1 is required for normal kidney development. RESULTS: Ablation of Esrp1 in mice, alone or together with its paralog Esrp2, was associated with reduced kidney size and increased incidence of renal aplasia. Three-dimensional imaging showed that embryonic Esrp1 knockout (KO) kidneys had fewer ureteric tips and reduced nephron numbers. Analysis of alternative splicing in Esrp-null ureteric epithelial cells by RNA-Seq confirmed a splicing switch in Fgfr2 as well as numerous other transcripts. CONCLUSIONS: Our findings reveal that Esrp1-regulated splicing in ureteric epithelial cells plays an important role in renal development. Defects in Esrp1 KO kidneys likely reflect reduced and/or absent ureteric branching, leading to decreased nephron induction secondary to incorrect Fgfr2 splicing and other splicing alterations. Developmental Dynamics 245:991-1000, 2016. © 2016 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.


Subject(s)
RNA-Binding Proteins/metabolism , Ureter/cytology , Ureter/metabolism , Animals , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Kidney/cytology , Kidney/embryology , Male , Mice , Mice, Knockout , Nephrons/cytology , Nephrons/metabolism , RNA Splicing/genetics , RNA Splicing/physiology , RNA-Binding Proteins/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/physiology
11.
Proc Natl Acad Sci U S A ; 109(47): 19280-5, 2012 Nov 20.
Article in English | MEDLINE | ID: mdl-23129656

ABSTRACT

Human mena (hMENA), a member of the actin cytoskeleton regulators Ena/VASP, is overexpressed in high-risk preneoplastic lesions and in primary breast tumors and has been identified as playing a role in invasiveness and poor prognosis in breast cancers that express HER2. Here we identify a unique isoform, hMENAΔv6, derived from the hMENA alternative splicing program. In an isogenic model of human breast cancer progression, we show that hMENA(11a) is expressed in premalignant cells, whereas hMENAΔv6 expression is restricted to invasive cancer cells. "Reversion" of the malignant phenotype leads to concurrent down-regulation of all hMENA isoforms. In breast cancer cell lines, isoform-specific hMENA overexpression or knockdown revealed that in the absence of hMENA(11a), overexpression of hMENAΔv6 increased cell invasion, whereas overexpression of hMENA(11a) reduced the migratory and invasive ability of these cells. hMENA(11a) splicing was shown to be dependent on the epithelial regulator of splicing 1 (ESRP1), and forced expression of ESRP1 in invasive mesenchymal breast cancer cells caused a phenotypic switch reminiscent of a mesenchymal-to-epithelial transition (MET) characterized by changes in the cytoskeletal architecture, reexpression of hMENA(11a), and a reduction in cell invasion. hMENA-positive primary breast tumors, which are hMENA(11a)-negative, are more frequently E-cadherin low in comparison with tumors expressing hMENA(11a). These data suggest that polarized and growth-arrested cellular architecture correlates with absence of alternative hMENA isoform expression, and that the hMENA splicing program is relevant to malignant progression in invasive disease.


Subject(s)
Alternative Splicing/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Mesoderm/pathology , Microfilament Proteins/genetics , Actin Cytoskeleton/metabolism , Antigens, CD , Cadherins/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cloning, Molecular , Disease Progression , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Mesoderm/metabolism , Microfilament Proteins/metabolism , Molecular Sequence Data , Neoplasm Invasiveness , Phenotype , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/metabolism , Transfection , Vimentin/metabolism
12.
EMBO J ; 29(19): 3286-300, 2010 Oct 06.
Article in English | MEDLINE | ID: mdl-20711167

ABSTRACT

Alternative splicing achieves coordinated changes in post-transcriptional gene expression programmes through the activities of diverse RNA-binding proteins. Epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) are cell-type-specific regulators of transcripts that switch splicing during the epithelial-mesenchymal transition (EMT). To define a comprehensive programme of alternative splicing that is regulated during the EMT, we identified an extensive ESRP-regulated splicing network of hundreds of alternative splicing events within numerous genes with functions in cell-cell adhesion, polarity, and migration. Loss of this global ESRP-regulated epithelial splicing programme induces the phenotypic changes in cell morphology that are observed during the EMT. Components of this splicing signature provide novel molecular markers that can be used to characterize the EMT. Bioinformatics and experimental approaches revealed a high-affinity ESRP-binding motif and a predictive RNA map that governs their activity. This work establishes the ESRPs as coordinators of a complex alternative splicing network that adds an important post-transcriptional layer to the changes in gene expression that underlie epithelial-mesenchymal transitions during development and disease.


Subject(s)
Alternative Splicing/physiology , Cell Differentiation/physiology , Epithelial Cells/cytology , Gene Expression Regulation/physiology , Mesoderm/cytology , RNA-Binding Proteins/physiology , Binding Sites/genetics , Cell Adhesion/genetics , Cell Line , Cell Movement/genetics , Cell Polarity/genetics , Computational Biology , Electrophoretic Mobility Shift Assay , Fluorescent Antibody Technique , Humans , Immunoblotting , Microarray Analysis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Vesicular Transport Proteins/genetics
13.
Nucleic Acids Res ; 40(8): e61, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22266656

ABSTRACT

Ultra-deep RNA sequencing has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We develop MATS (multivariate analysis of transcript splicing), a bayesian statistical framework for flexible hypothesis testing of differential alternative splicing patterns on RNA-Seq data. MATS uses a multivariate uniform prior to model the between-sample correlation in exon splicing patterns, and a Markov chain Monte Carlo (MCMC) method coupled with a simulation-based adaptive sampling procedure to calculate the P-value and false discovery rate (FDR) of differential alternative splicing. Importantly, the MATS approach is applicable to almost any type of null hypotheses of interest, providing the flexibility to identify differential alternative splicing events that match a given user-defined pattern. We evaluated the performance of MATS using simulated and real RNA-Seq data sets. In the RNA-Seq analysis of alternative splicing events regulated by the epithelial-specific splicing factor ESRP1, we obtained a high RT-PCR validation rate of 86% for differential exon skipping events with a MATS FDR of <10%. Additionally, over the full list of RT-PCR tested exons, the MATS FDR estimates matched well with the experimental validation rate. Our results demonstrate that MATS is an effective and flexible approach for detecting differential alternative splicing from RNA-Seq data.


Subject(s)
Alternative Splicing , High-Throughput Nucleotide Sequencing , Sequence Analysis, RNA , Bayes Theorem , Brain/metabolism , Cell Line, Tumor , Humans , Multivariate Analysis , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction
14.
Adv Exp Med Biol ; 825: 267-302, 2014.
Article in English | MEDLINE | ID: mdl-25201109

ABSTRACT

The epithelial to mesenchymal transition (EMT) and reverse mesenchymal to epithelial transition (MET) are developmentally conserved processes that are essential for patterning of developing embryos and organs. The EMT/MET are further utilized in wound healing, but they can also be hijacked by cancer cells to promote tumor progression and metastasis. The molecular pathways governing these processes have historically focused on the transcriptional regulation and networks that control them. Indeed, global profiling of transcriptional changes has provided a wealth of information into how these networks are regulated, the downstream targets, and functional consequence of alterations to the global transcriptome. However, recent evidence has revealed that the posttranscriptional landscape of the cell is also dramatically altered during the EMT/MET and contributes to changes in cell behavior and phenotypes. While studies of this aspect of EMT biology are still in their infancy, recent progress has been achieved by the identification of several RNA binding proteins (RBPs) that regulate splicing, polyadenylation, mRNA stability, and translational control during EMT. This chapter focuses on the global impact of RBPs that regulate mRNA maturation as well as outlines the functional impact of several key posttranscriptional changes during the EMT. The growing evidence of RBP involvement in the cellular transformation during EMT underscores that a coordinated regulation of both transcriptional and posttranscriptional changes is essential for EMT. Furthermore, new discoveries into these events will paint a more detailed picture of the transcriptome during the EMT/MET and provide novel molecular targets for treatment of human diseases.


Subject(s)
Epithelial-Mesenchymal Transition/physiology , Genome, Human/physiology , RNA Processing, Post-Transcriptional/physiology , RNA-Binding Proteins , Transcriptome/physiology , Animals , Gene Expression Profiling/methods , Genome-Wide Association Study/methods , Humans , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism
15.
Semin Cancer Biol ; 22(5-6): 417-27, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22548723

ABSTRACT

The epithelial-to-mesenchymal transition (EMT) is an important developmental process that is also implicated in disease pathophysiology, such as cancer progression and metastasis. A wealth of literature in recent years has identified important transcriptional regulators and large-scale changes in gene expression programs that drive the phenotypic changes that occur during the EMT. However, in the past couple of years it has become apparent that extensive changes in alternative splicing also play a profound role in shaping the changes in cell behavior that characterize the EMT. While long known splicing switches in FGFR2 and p120-catenin provided hints of a larger program of EMT-associated alternative splicing, the recent identification of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) began to reveal this genome-wide post-transcriptional network. Several studies have now demonstrated the truly vast extent of this alternative splicing program. The global switches in splicing associated with the EMT add an important additional layer of post-transcriptional control that works in harmony with transcriptional and epigenetic regulation to effect complex changes in cell shape, polarity, and behavior that mediate transitions between epithelial and mesenchymal cell states. Future challenges include the need to investigate the functional consequences of these splicing switches at both the individual gene as well as systems level.


Subject(s)
Alternative Splicing , Epithelial-Mesenchymal Transition/genetics , RNA Precursors/genetics , RNA, Messenger/metabolism , Animals , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Humans , RNA Isoforms/genetics , RNA Isoforms/metabolism , RNA Precursors/metabolism
16.
bioRxiv ; 2024 Jul 09.
Article in English | MEDLINE | ID: mdl-39026848

ABSTRACT

Hepatocyte polyploidy and maturity are critical to acquiring specialized liver functions. Multiple intra- and extracellular factors influence ploidy, but how they cooperate temporally to steer liver polyploidization and maturation or how post-transcriptional mechanisms integrate into these paradigms is unknown. Here, we identified an important regulatory hierarchy in which postnatal activation of Epithelial-Splicing-Regulatory-Protein-2 (ESRP2) stimulates biogenesis of liver-specific microRNA (miR-122), thereby facilitating polyploidization, maturation, and functional competence of hepatocytes. By determining transcriptome-wide protein-RNA interactions in vivo and integrating them with single-cell and bulk hepatocyte RNA-seq datasets, we delineate an ESRP2-driven RNA processing program that drives sequential replacement of fetal-to-adult transcript isoforms. Specifically, ESRP2 binds the primary miR-122 host gene transcript to promote its processing/biogenesis. Combining constitutive and inducible ESRP2 gain- and loss-of-function mice models with miR-122 rescue experiments, we demonstrate that timed activation of ESRP2 augments miR-122-driven program of cytokinesis failure, ensuring proper onset and extent of hepatocyte polyploidization.

17.
bioRxiv ; 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-39005284

ABSTRACT

Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp - Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.

18.
iScience ; 25(10): 105205, 2022 Oct 21.
Article in English | MEDLINE | ID: mdl-36238894

ABSTRACT

The epithelial splicing regulatory proteins, ESRP1 and ESRP2, are essential for mammalian development through the regulation of a global program of alternative splicing of genes involved in the maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1, we performed enhanced crosslinking immunoprecipitation coupled with high-throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to promote either exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3' and 5' untranslated regions (UTRs) of genes involved in epithelial cell function, suggesting that its post-transcriptional functions extend beyond splicing regulation.

19.
Bioinformatics ; 26(2): 268-9, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-19933160

ABSTRACT

MOTIVATION: The Affymetrix Human Exon Junction Array is a newly designed high-density exon-sensitive microarray for global analysis of alternative splicing. Contrary to the Affymetrix exon 1.0 array, which only contains four probes per exon and no probes for exon-exon junctions, this new junction array averages eight probes per probeset targeting all exons and exon-exon junctions observed in the human mRNA/EST transcripts, representing a significant increase in the probe density for alternative splicing events. Here, we present MADS+, a computational pipeline to detect differential splicing events from the Affymetrix exon junction array data. For each alternative splicing event, MADS+ evaluates the signals of probes targeting competing transcript isoforms to identify exons or splice sites with different levels of transcript inclusion between two sample groups. MADS+ is used routinely in our analysis of Affymetrix exon junction arrays and has a high accuracy in detecting differential splicing events. For example, in a study of the novel epithelial-specific splicing regulator ESRP1, MADS+ detects hundreds of exons whose inclusion levels are dependent on ESRP1, with a RT-PCR validation rate of 88.5% (153 validated out of 173 tested). AVAILABILITY: MADS+ scripts, documentations and annotation files are available at http://www.medicine.uiowa.edu/Labs/Xing/MADSplus/.


Subject(s)
Alternative Splicing/genetics , Exons , Oligonucleotide Array Sequence Analysis/standards , Software , Humans , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNA/methods
20.
Mol Cell Biol ; 27(9): 3290-302, 2007 May.
Article in English | MEDLINE | ID: mdl-17325043

ABSTRACT

Posttranscriptional controls, mediated primarily by RNA-protein complexes, have the potential to alter multiple steps in RNA processing and function. Human alpha-globin mRNA is bound at a C-rich motif in the 3' untranslated region (3'UTR) by the KH domain protein alpha-globin poly(C)-binding protein (alphaCP). This "alpha-complex" is essential to cytoplasmic stability of alpha-globin mRNA in erythroid cells. Here we report that the 3'UTR alpha-complex also serves an independent nuclear role as a splice enhancer. Consistent with this role, we find that alphaCP binds alpha-globin transcripts prior to splicing. Surprisingly, this binding occurs at C-rich sites within intron I as well as at the 3'UTR C-rich determinant. The intronic and 3'UTR alphaCP complexes appear to have distinct effects on splicing. While intron I complexes repress intron I excision, the 3'UTR complex enhances splicing of the full-length transcript both in vivo and in vitro. In addition to its importance to splicing, nuclear assembly of the 3'UTR alphaCP complex may serve to "prepackage" alpha-globin mRNA with its stabilizing complex prior to cytoplasmic export. Linking nuclear and cytoplasmic controls by the action of a particular RNA-binding protein, as reported here, may represent a modality of general importance in eukaryotic gene regulation.


Subject(s)
3' Untranslated Regions/genetics , Cell Nucleus/genetics , Globins/genetics , RNA Splicing/genetics , RNA Stability/genetics , Regulatory Sequences, Ribonucleic Acid/genetics , 3' Untranslated Regions/metabolism , Cell Line, Tumor , Humans , Introns/genetics , Molecular Sequence Data , Protein Binding , RNA-Binding Proteins/metabolism , Transcription, Genetic/genetics
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