ABSTRACT
Lipid nanoparticles (LNPs) are widely used for mRNA delivery, with cationic lipids greatly affecting biodistribution, cellular uptake, endosomal escape and transfection efficiency. However, the laborious synthesis of cationic lipids limits the discovery of efficacious candidates and slows down scale-up manufacturing. Here we develop a one-pot, tandem multi-component reaction based on the rationally designed amine-thiol-acrylate conjugation, which enables fast (1 h) and facile room-temperature synthesis of amidine-incorporated degradable (AID) lipids. Structure-activity relationship analysis of a combinatorial library of 100 chemically diverse AID-lipids leads to the identification of a tail-like amine-ring-alkyl aniline that generally affords efficacious lipids. Experimental and theoretical studies show that the embedded bulky benzene ring can enhance endosomal escape and mRNA delivery by enabling the lipid to adopt a more conical shape. The lead AID-lipid can not only mediate local delivery of mRNA vaccines and systemic delivery of mRNA therapeutics, but can also alter the tropism of liver-tropic LNPs to selectively deliver gene editors to the lung and mRNA vaccines to the spleen.
ABSTRACT
The ionizable lipidoid is a key component of lipid nanoparticles (LNPs). Degradable lipidoids containing extended alkyl branches have received tremendous attention, yet their optimization and investigation are underappreciated. Here, we devise an in situ construction method for the combinatorial synthesis of degradable branched (DB) lipidoids. We find that appending branch tails to inefficacious lipidoids via degradable linkers boosts mRNA delivery efficiency up to three orders of magnitude. Combinatorial screening and systematic investigation of two libraries of DB-lipidoids reveal important structural criteria that govern their in vivo potency. The lead DB-LNP demonstrates robust delivery of mRNA therapeutics and gene editors into the liver. In a diet-induced obese mouse model, we show that repeated administration of DB-LNP encapsulating mRNA encoding human fibroblast growth factor 21 alleviates obesity and fatty liver. Together, we offer a construction strategy for high-throughput and cost-efficient synthesis of DB-lipidoids. This study provides insights into branched lipidoids for efficient mRNA delivery.
Subject(s)
Nanoparticles , Animals , Mice , Humans , RNA, Messenger/genetics , Nanoparticles/chemistry , RNA, Small InterferingABSTRACT
Ornithine transcarbamylase deficiency is a rare X-linked genetic urea cycle disorder leading to episodes of acute hyperammonemia, adverse cognitive and neurological effects, hospitalizations, and in some cases death. DTX301, a non-replicating, recombinant self-complimentary adeno-associated virus vector serotype 8 (scAAV8)-encoding human ornithine transcarbamylase, is a promising gene therapy for ornithine transcarbamylase deficiency; however, the impact of sex and prophylactic immunosuppression on ornithine transcarbamylase gene therapy outcomes is not well characterized. This study sought to describe the impact of sex and immunosuppression in adult, sexually mature female and male cynomolgus macaques through day 140 after DTX301 administration. Four study groups (n = 3/group) were included: male non-immunosuppressed; male immunosuppressed; female non-immunosuppressed; and female immunosuppressed. DTX301 was well tolerated with and without immunosuppression; no notable differences were observed between female and male groups across outcome measures. Prednisolone-treated animals exhibited a trend toward greater vector genome and transgene expression, although the differences were not statistically significant. The hepatic interferon gene signature was significantly decreased in prednisolone-treated animals, and a significant inverse relationship was observed between interferon gene signature levels and hepatic vector DNA and transgene RNA. These observations were not sustained upon immunosuppression withdrawal. Further studies may determine whether the observed effect can be prolonged.
ABSTRACT
A major challenge to advance lipid nanoparticles (LNPs) for RNA therapeutics is the development of formulations that can be produced reliably across the various scales of drug development. Microfluidics can generate LNPs with precisely defined properties, but have been limited by challenges in scaling throughput. To address this challenge, we present a scalable, parallelized microfluidic device (PMD) that incorporates an array of 128 mixing channels that operate simultaneously. The PMD achieves a >100× production rate compared to single microfluidic channels, without sacrificing desirable LNP physical properties and potency typical of microfluidic-generated LNPs. In mice, we show superior delivery of LNPs encapsulating either Factor VII siRNA or luciferase-encoding mRNA generated using a PMD compared to conventional mixing, with a 4-fold increase in hepatic gene silencing and 5-fold increase in luciferase expression, respectively. These results suggest that this PMD can generate scalable and reproducible LNP formulations needed for emerging clinical applications, including RNA therapeutics and vaccines.
Subject(s)
Lab-On-A-Chip Devices , Nanoparticles , Animals , Lipids , Mice , RNA, Messenger , RNA, Small Interfering/geneticsABSTRACT
Gene disruption via programmable, sequence-specific nucleases represents a promising gene therapy strategy in which the reduction of specific protein levels provides a therapeutic benefit. Proprotein convertase subtilisin/kexin type 9 (PCSK9), an antagonist of the low-density lipoprotein (LDL) receptor, is a suitable target for nuclease-mediated gene disruption as an approach to treat hypercholesterolemia. We sought to determine the long-term durability and safety of PCSK9 knockdown in non-human primate (NHP) liver by adeno-associated virus (AAV)-delivered meganuclease following our initial report on the feasibility of this strategy. Six previously treated NHPs and additional NHPs administered AAV-meganuclease in combination with corticosteroid treatment or an alternative AAV serotype were monitored for a period of up to 3 years. The treated NHPs exhibited a sustained reduction in circulating PCSK9 and LDL cholesterol (LDL-c) through the course of the study concomitant with stable gene editing of the PCSK9 locus. Low-frequency off-target editing remained stable, and no obvious adverse changes in histopathology of the liver were detected. We demonstrate similar on-target nuclease activity in primary human hepatocytes using a chimeric liver-humanized mouse model. These studies demonstrate that targeted in vivo gene disruption exerts a lasting therapeutic effect and provide pivotal data for safety considerations, which support clinical translation.
Subject(s)
Gene Editing , Lipoproteins, LDL/metabolism , Proprotein Convertase 9/genetics , Animals , CRISPR-Cas Systems , Dependovirus/genetics , Disease Models, Animal , Gene Expression , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors/genetics , Lipoproteins, LDL/genetics , Liver/metabolism , Mice , Mice, Knockout , Primates , Proprotein Convertase 9/metabolismABSTRACT
Nucleic acids, such as messenger RNAs, antisense oligonucleotides, and short interfering RNAs, hold great promise for treating previously 'undruggable' diseases. However, there are numerous biological barriers that hinder nucleic acid delivery to target cells and tissues. While lipid nanoparticles (LNPs) have been developed to protect nucleic acids from degradation and mediate their intracellular delivery, it is challenging to predict how alterations in LNP formulation parameters influence delivery to different organs. In this study, we utilized high-throughput in vivo screening to probe for structure-function relationships of intravenously administered LNPs along with quartz crystal microbalance with dissipation monitoring (QCM-D) to measure the binding affinity of LNPs to apolipoprotein E (ApoE), a protein implicated in the clearance and uptake of lipoproteins by the liver. High-throughput in vivo screening of a library consisting of 96 LNPs identified several formulations containing the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) that preferentially accumulated in the liver, while identical LNPs that substituted DOPE with the helper lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) preferentially accumulated in the spleen. Using QCM-D, it was found that one DOPE-containing LNP formulation (LNP 42) had stronger interactions with ApoE than an identical LNP formulation that substituted DOPE with DSPC (LNP 90). In order to further validate our findings, we formulated LNP 42 and LNP 90 to encapsulate Cy3-siRNA or mRNA encoding for firefly luciferase. The DSPC-containing LNP (LNP 90) was found to increase delivery to the spleen for both siRNA (two-fold) and mRNA (five-fold). In terms of liver delivery, the DOPE-containing LNP (LNP 42) enhanced mRNA delivery to the liver by two-fold and improved liver transfection by three-fold. Understanding the role of the helper lipid in LNP biodistribution and ApoE adsorption may aid in the future design of LNPs for nucleic acid therapeutics.
Subject(s)
Nanoparticles , Spleen , Adsorption , Lipids , Liver/metabolism , RNA, Small Interfering/metabolism , Tissue DistributionABSTRACT
Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Cell Self Renewal , DNA-Binding Proteins/physiology , LIM Domain Proteins/physiology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Thymocytes/cytology , Adaptor Proteins, Signal Transducing/genetics , Adoptive Transfer , Animals , Antigens, CD/biosynthesis , Cell Transformation, Neoplastic , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Gene Deletion , Gene Knock-In Techniques , LIM Domain Proteins/deficiency , LIM Domain Proteins/genetics , Lymphopoiesis , Mice , Mice, Inbred C57BL , Mice, Transgenic , Proto-Oncogene Mas , RNA-Seq , Radiation Chimera , Radiation Tolerance , Thymocytes/metabolism , Thymocytes/radiation effects , Thymocytes/transplantationABSTRACT
HIRA is a histone chaperone that deposits the histone variant H3.3 in transcriptionally active genes. In DiGeorge syndromes, a DNA stretch encompassing HIRA is deleted. The syndromes manifest varied abnormalities, including immunodeficiency and thrombocytopenia. HIRA is essential in mice, as total knockout (KO) results in early embryonic death. However, the role of HIRA in hematopoiesis is poorly understood. We investigate hematopoietic cell-specific Hira deletion in mice and show that it dramatically reduces bone marrow hematopoietic stem cells (HSCs), resulting in anemia, thrombocytopenia, and lymphocytopenia. In contrast, fetal hematopoiesis is normal in Hira-KO mice, although fetal HSCs lack the reconstitution capacity. Transcriptome analysis reveals that HIRA is required for expression of many transcription factors and signaling molecules critical for HSCs. ATAC-seq analysis demonstrates that HIRA establishes HSC-specific DNA accessibility, including the SPIB/PU.1 sites. Together, HIRA provides a chromatin environment essential for HSCs, thereby steering their development and survival.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin/metabolism , DiGeorge Syndrome/genetics , Histone Chaperones/metabolism , Transcription Factors/metabolism , Animals , Cell Cycle Proteins/genetics , Chromatin/genetics , DiGeorge Syndrome/metabolism , Female , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Histone Chaperones/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Transcription Factors/geneticsABSTRACT
THEMIS, a T cell-specific protein with high expression in CD4+CD8+ thymocytes, has a crucial role in positive selection and T cell development. THEMIS lacks defined catalytic domains but contains two tandem repeats of a distinctive module of unknown function (CABIT). Here we found that THEMIS directly regulated the catalytic activity of the tyrosine phosphatase SHP-1. This action was mediated by the CABIT modules, which bound to the phosphatase domain of SHP-1 and promoted or stabilized oxidation of SHP-1's catalytic cysteine residue, which inhibited the tyrosine-phosphatase activity of SHP-1. Deletion of SHP-1 alleviated the developmental block in Themis-/- thymocytes. Thus, THEMIS facilitates thymocyte positive selection by enhancing the T cell antigen receptor signaling response to low-affinity ligands.
Subject(s)
Clonal Selection, Antigen-Mediated/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Line , Gene Deletion , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Knockout , Oxidation-Reduction , Protein Binding , Protein Interaction Domains and Motifs , Protein Tyrosine Phosphatase, Non-Receptor Type 6/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 6/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Reactive Oxygen Species/metabolism , T-Lymphocytes/cytology , Thymocytes/cytology , Thymocytes/immunology , Thymocytes/metabolismABSTRACT
The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B-cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.
Subject(s)
B-Lymphocytes/physiology , Clonal Selection, Antigen-Mediated , Germinal Center/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation , Adaptive Immunity , Animals , Cell Differentiation , Cell Lineage , Cells, Cultured , GRB2 Adaptor Protein/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Phospholipase C gamma/metabolism , Receptors, Antigen, B-Cell/metabolism , Self Tolerance , src-Family Kinases/metabolismABSTRACT
The epithelial-to-mesenchymal transition (EMT) is an essential biological process during embryonic development that is also implicated in cancer metastasis. While the transcriptional regulation of EMT has been well studied, the role of alternative splicing (AS) regulation in EMT remains relatively uncharacterized. We previously showed that the epithelial cell-type-specific proteins epithelial splicing regulatory proteins 1 (ESRP1) and ESRP2 are important for the regulation of many AS events that are altered during EMT. However, the contributions of the ESRPs and other splicing regulators to the AS regulatory network in EMT require further investigation. Here, we used a robust in vitro EMT model to comprehensively characterize splicing switches during EMT in a temporal manner. These investigations revealed that the ESRPs are the major regulators of some but not all AS events during EMT. We determined that the splicing factor RBM47 is downregulated during EMT and also regulates numerous transcripts that switch splicing during EMT. We also determined that Quaking (QKI) broadly promotes mesenchymal splicing patterns. Our study highlights the broad role of posttranscriptional regulation during the EMT and the important role of combinatorial regulation by different splicing factors to fine tune gene expression programs during these physiological and developmental transitions.
Subject(s)
Alternative Splicing , Epithelial-Mesenchymal Transition , Gene Regulatory Networks , Neoplasms/genetics , Cell Line, Tumor , Down-Regulation , Humans , Models, Biological , RNA-Binding Proteins/genetics , Sequence Analysis, RNAABSTRACT
Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development. Mice with ablation of Epithelial splicing regulatory protein (Esrp1) develop cleft lip and palate. Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease. Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis. We profiled the global Esrp-mediated splicing regulatory program in epidermis, which revealed large-scale programs of epithelial cell-type-specific splicing required for epithelial cell functions. These mice represent a valuable model for evaluating the essential role for AS in development and function of epithelial cells, which play essential roles in tissue homeostasis in numerous organs, and provide a genetic tool to evaluate important functional properties of epithelial-specific splice variants in vivo.
Subject(s)
Animal Structures/embryology , Epithelial Cells/physiology , RNA-Binding Proteins/metabolism , Animals , Female , Gene Deletion , Gene Expression Profiling , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , RNA-Binding Proteins/geneticsABSTRACT
Elucidation of the genetic pathways that control red blood cell development has been a central goal of erythropoiesis research over the past decade. Notably, data from several recent studies have provided new insights into the regulation of erythroid gene transcription. Transcription profiling demonstrates that erythropoiesis is mainly controlled by a small group of lineage-restricted transcription factors [Gata binding protein 1 (Gata1), T cell acute lymphocytic leukemia 1 protein (Tal1), and Erythroid Kruppel-like factor (EKLF; henceforth referred to as Klf1)]. Binding-site mapping using ChIP-Seq indicates that most DNA-bound Gata1 and Tal1 proteins are contained within higher order complexes (Ldb1 complexes) that include the nuclear adapters Ldb1 and Lmo2. Ldb1 complexes regulate Klf1, and Ldb1 complex-binding sites frequently colocalize with Klf1 at erythroid genes and cis-regulatory elements, indicating strong functional synergy between Gata1, Tal1, and Klf1. Together with new data demonstrating that Ldb1 can mediate long-range promoter-enhancer interactions, these findings provide a foundation for the first comprehensive models of the global regulation of erythroid gene transcription.
Subject(s)
Erythropoiesis/genetics , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epigenetic Repression , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Gene Expression Regulation , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , LIM Domain Proteins/genetics , LIM Domain Proteins/metabolism , Mice , Models, Animal , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Sequence Analysis, DNA , T-Cell Acute Lymphocytic Leukemia Protein 1 , Transcription Factors/geneticsABSTRACT
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/metabolismABSTRACT
Alternative splicing of pre-mRNA transcripts is a critical and extensively utilized mechanism of gene regulation. In this chapter, we describe a series of fluorescent and luminescent minigene reporters our lab has used to facilitate the study of alternative splicing regulation in cultured cells. Through the use of different versions of these minigenes, the inclusion level of a cassette exon can be directly ascertained by fluorescence or luciferase activity, thereby making it possible to establish cell-based assays for induced exon splicing or skipping. A successful application of these minigenes in a high-throughput cDNA screen led to the identification of a cell type-specific regulator of FGFR2 splicing, illustrating the power of these reporters to yield novel insights into alternative splicing. The methods and minigenes described are adaptable for genetic screens to uncover novel regulators of a broader set of alternative splicing events in other gene transcripts. These reporters also have a dynamic range that is suitable for small molecule screening for compounds that can regulate splicing.
Subject(s)
Alternative Splicing , Exons , Gene Library , Genes, Reporter , High-Throughput Screening Assays/methods , Animals , Base Sequence , Cell Line , Cells, Cultured , Flow Cytometry/methods , Humans , Introns , Luciferases/genetics , Luminescent Agents/metabolism , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA/genetics , RNA/isolation & purification , RNA Precursors/genetics , RNA Precursors/isolation & purification , TransfectionABSTRACT
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/geneticsABSTRACT
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/methodsABSTRACT
Cell-type and tissue-specific alternative splicing events are regulated by combinatorial control involving both abundant RNA binding proteins as well as those with more discrete expression and specialized functions. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are recently discovered epithelial-specific RNA binding proteins that promote splicing of the epithelial variant of the FGFR2, ENAH, CD44 and CTNND1 transcripts. To catalogue a larger set of splicing events under the regulation of the ESRPs we profiled splicing changes induced by RNA interference-mediated knockdown of ES RP1 and ES RP2 expression in a human epithelial cell line using the splicing sensitive Affymetrix Exon ST1.0 Arrays. Analysis of the microarray data resulted in the identification of over a hundred candidate ESRP regulated splicing events. We were able to independently validate 38 of these targets by RT-PCR. The ESRP regulated events encompass all known types of alternative splicing events, most prominent being alternative cassette exons and splicing events leading to alternative 3' terminal exons. Importantly, a number of these regulated splicing events occur in gene transcripts that encode proteins with well-described roles in the regulation of actin cytoskeleton organization, cell-cell adhesion, cell polarity and cell migration. In sum, this work reveals a novel list of transcripts differentially spliced in epithelial and mesenchymal cells, implying that coordinated alternative splicing plays a critical role in determination of cell type identity. These results further establish ESRP1 and ESRP2 as global regulators of an epithelial splicing regulatory network.
Subject(s)
Alternative Splicing , RNA-Binding Proteins/genetics , Cells, Cultured , Epithelial Cells , Humans , Mesenchymal Stem Cells , Oligonucleotide Array Sequence Analysis , RNA, MessengerABSTRACT
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.