ABSTRACT
Alternative splicing (AS) patterns have diverged rapidly during vertebrate evolution, yet the functions of most species- and lineage-specific splicing events are not known. We observe that mammalian-specific AS events are enriched in transcript sequences encoding intrinsically disordered regions (IDRs) of proteins, in particular those containing glycine/tyrosine repeats that mediate formation of higher-order protein assemblies implicated in gene regulation and human disease. These evolutionary changes impact nearly all members of the hnRNP A and D families of RNA binding proteins. Regulation of these events requires formation of unusual, long-range mammalian-specific RNA duplexes. Differential inclusion of the alternative exons controls the formation of tyrosine-dependent multivalent hnRNP assemblies that, in turn, function to globally regulate splicing. Together, our results demonstrate that AS control of IDR-mediated interactions between hnRNPs represents an important and recurring mechanism underlying splicing regulation. Furthermore, this mechanism has expanded the regulatory capacity of mammalian cells.
Subject(s)
Alternative Splicing , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Mammals/genetics , Amino Acid Sequence , Animals , Gene Expression Regulation , Humans , Mammals/metabolism , Protein Isoforms/metabolism , RNA Precursors/metabolism , Sequence Alignment , Vertebrates/genetics , Vertebrates/metabolismABSTRACT
Efficient targeted control of splicing is a major goal of functional genomics and therapeutic applications. Guide (g)RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility of these systems. However, efficient, specific, and generalizable activation of endogenous exons using this approach has not been previously reported. By screening over 300 dCasRx-splicing factor fusion proteins tethered to splicing reporters, we identify dCasRx-RBM25 as a potent activator of exons. Moreover, dCasRx-RBM25 efficiently activates the splicing of â¼90% of targeted endogenous alternative exons and displays high on-target specificity. Using gRNA arrays for combinatorial targeting, we demonstrate that dCasRx-RBM25 enables multiplexed activation and repression of exons. Using this feature, the targeting of neural-regulated exons in Ptpb1 and Puf60 in embryonic stem cells reveals combinatorial effects on downstream alternative splicing events controlled by these factors. Collectively, our results enable versatile, combinatorial exon-resolution functional assays and splicing-directed therapeutic applications.
Subject(s)
Alternative Splicing , CRISPR-Cas Systems , Exons , RNA Splicing Factors , RNA-Binding Proteins , Humans , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , HEK293 Cells , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , RNA, Guide, CRISPR-Cas Systems/genetics , RNA, Guide, CRISPR-Cas Systems/metabolism , Animals , MiceABSTRACT
Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.
Subject(s)
Alternative Splicing , RNA Splicing , Exons/genetics , Introns , RNAABSTRACT
Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.
Subject(s)
Poly A , Polyadenylation , 3' Untranslated Regions , Humans , Poly A/metabolism , RNA, Messenger/metabolism , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Zinc/metabolismABSTRACT
The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.
Subject(s)
Cell Nucleus , RNA-Binding Proteins , Cell Nucleus/genetics , Cell Nucleus/metabolism , Gene Expression Regulation , Humans , Introns/genetics , RNA Splicing , RNA-Binding Proteins/geneticsABSTRACT
Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.
Subject(s)
Neurogenesis , RNA Splicing , Alternative Splicing , Animals , Exons/genetics , Mammals , Mice , Neurogenesis/genetics , Neurons , RNA-Binding Proteins/geneticsABSTRACT
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.
Subject(s)
Alternative Splicing , Child Development Disorders, Pervasive/pathology , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Animals , Child Development Disorders, Pervasive/metabolism , Humans , Mice , Models, Molecular , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Neurogenesis , Protein Interaction Domains and Motifs , Sequence Analysis, RNA , Temporal Lobe/pathologyABSTRACT
Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport, and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive crosstalk between gene regulatory layers takes advantage of dynamic spatial, physical, and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control.
Subject(s)
Gene Regulatory Networks , RNA Splicing , Alternative Splicing , Animals , Cell Nucleus/genetics , Chromatin/metabolism , Humans , Regulatory Sequences, Ribonucleic Acid , Transcription, GeneticABSTRACT
Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.
Subject(s)
Autistic Disorder/physiopathology , Cognitive Dysfunction/pathology , Eukaryotic Initiation Factor-4G/physiology , Exons/genetics , Fragile X Mental Retardation Protein/metabolism , Neuroblastoma/pathology , Neurons/pathology , Animals , Behavior, Animal , Cognitive Dysfunction/genetics , Cognitive Dysfunction/metabolism , Fragile X Mental Retardation Protein/genetics , Male , Mice , Mice, Inbred C57BL , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neurogenesis , Neurons/metabolism , Protein Biosynthesis , RNA Splicing , Tumor Cells, CulturedABSTRACT
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.
Subject(s)
Alveolar Epithelial Cells/metabolism , COVID-19/metabolism , Phosphoproteins/metabolism , Proteome/metabolism , SARS-CoV-2/metabolism , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , Antiviral Agents , COVID-19/genetics , COVID-19/pathology , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Cytoskeleton , Drug Evaluation, Preclinical , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Induced Pluripotent Stem Cells/virology , Phosphoproteins/genetics , Protein Transport , Proteome/genetics , SARS-CoV-2/genetics , Signal Transduction , Vero Cells , COVID-19 Drug TreatmentABSTRACT
High-throughput sequencing-based methods and their applications in the study of transcriptomes have revolutionized our understanding of alternative splicing. Networks of functionally coordinated and biologically important alternative splicing events continue to be discovered in an ever-increasing diversity of cell types in the context of physiologically normal and disease states. These studies have been complemented by efforts directed at defining sequence codes governing splicing and their cognate trans-acting factors, which have illuminated important combinatorial principles of regulation. Additional studies have revealed critical roles of position-dependent, multivalent protein-RNA interactions that direct splicing outcomes. Investigations of evolutionary changes in RNA binding proteins, splice variants, and associated cis elements have further shed light on the emergence, mechanisms, and functions of splicing networks. Progress in these areas has emphasized the need for a coordinated, community-based effort to systematically address the functions of individual splice variants associated with normal and disease biology.
Subject(s)
Alternative Splicing/physiology , Evolution, Molecular , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , HumansABSTRACT
Cell-matrix interactions in 3D environments significantly differ from those in 2D cultures. As such, mechanisms of mechanotransduction in 2D cultures are not necessarily applicable to cell-encapsulating hydrogels that resemble features of tissue architecture. Accordingly, the characterization of molecular pathways in 3D matrices is expected to uncover insights into how cells respond to their mechanical environment in physiological contexts, and potentially also inform hydrogel-based strategies in cell therapies. In this study, a bone marrow-mimetic hydrogel was employed to systematically investigate the stiffness-responsive transcriptome of mesenchymal stromal cells. High matrix rigidity impeded integrin-collagen adhesion, resulting in changes in cell morphology characterized by a contractile network of actin proximal to the cell membrane. This resulted in a suppression of extracellular matrix-regulatory genes involved in the remodeling of collagen fibrils, as well as the upregulation of secreted immunomodulatory factors. Moreover, an investigation of long noncoding RNAs revealed that the cytoskeleton regulator RNA (CYTOR) contributes to these 3D stiffness-driven changes in gene expression. Knockdown of CYTOR using antisense oligonucleotides enhanced the expression of numerous mechanoresponsive cytokines and chemokines to levels exceeding those achievable by modulating matrix stiffness alone. Taken together, our findings further our understanding of mechanisms of mechanotransduction that are distinct from canonical mechanotransductive pathways observed in 2D cultures.
Subject(s)
Extracellular Matrix , Mechanotransduction, Cellular , Mesenchymal Stem Cells , RNA, Long Noncoding , Humans , Mesenchymal Stem Cells/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Extracellular Matrix/metabolism , Hydrogels/chemistry , Gene Expression Regulation , Collagen/metabolism , Cells, Cultured , Immunomodulation/geneticsABSTRACT
[This corrects the article DOI: 10.1371/journal.ppat.1008307.].
ABSTRACT
Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.
Subject(s)
Alternative Splicing , Cellular Reprogramming , Embryonic Stem Cells/metabolism , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental , Pluripotent Stem Cells/metabolism , Repressor Proteins/metabolism , Animals , DNA/metabolism , Embryonic Stem Cells/cytology , Genes, Homeobox , Humans , Mice , Pluripotent Stem Cells/cytology , Protein Isoforms/metabolismABSTRACT
Alternative splicing (AS) is a widespread process underlying the generation of transcriptomic and proteomic diversity and is frequently misregulated in human disease. Accordingly, an important goal of biomedical research is the development of tools capable of comprehensively, accurately, and efficiently profiling AS. Here, we describe Whippet, an easy-to-use RNA-seq analysis method that rapidly-with hardware requirements compatible with a laptop-models and quantifies AS events of any complexity without loss of accuracy. Using an entropic measure of splicing complexity, Whippet reveals that one-third of human protein coding genes produce transcripts with complex AS events involving co-expression of two or more principal splice isoforms. We observe that high-entropy AS events are more prevalent in tumor relative to matched normal tissues and correlate with increased expression of proto-oncogenic splicing factors. Whippet thus affords the rapid and accurate analysis of AS events of any complexity, and as such will facilitate future biomedical research.
Subject(s)
Alternative Splicing/genetics , Proteomics , RNA Splicing/genetics , Sequence Analysis, RNA/methods , Gene Expression Profiling/methods , Humans , Molecular Sequence Annotation , RNA, Messenger/genetics , TranscriptomeABSTRACT
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.
Subject(s)
Alternative Splicing/physiology , Genetic Engineering/methods , RNA Splice Sites/physiology , Animals , Autistic Disorder/genetics , CRISPR-Cas Systems/genetics , Cell Line , Exons/physiology , Humans , Mice , Nerve Tissue Proteins , Neurogenesis , Neurons , RNA Precursors/physiology , RNA Splicing/physiology , RNA-Binding Proteins , Ribonucleoproteins , Serine-Arginine Splicing Factors , SpliceosomesABSTRACT
Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.
Subject(s)
CRISPR-Cas Systems , Genes, Reporter , RNA Precursors , mRNA Cleavage and Polyadenylation Factors , Humans , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/genetics , Genome, Human , HEK293 Cells , mRNA Cleavage and Polyadenylation Factors/metabolism , mRNA Cleavage and Polyadenylation Factors/genetics , Polyadenylation , RNA Cleavage , RNA Polymerase II/metabolism , RNA Precursors/metabolism , RNA Precursors/geneticsABSTRACT
Heredity has a major role in autism spectrum disorder (ASD), yet underlying causal genetic variants have been defined only in a fairly small subset of cases. The enormous genetic heterogeneity associated with ASD emphasizes the importance of identifying convergent pathways and molecular mechanisms that are responsible for this disorder. We review how recent transcriptomic analyses have transformed our understanding of pathway convergence in ASD. In particular, deep RNA sequencing coupled with downstream investigations has revealed that a substantial fraction of autistic brains possess distinct transcriptomic signatures. These signatures are in part a consequence of altered neuronal activity and have a particular impact on pre-mRNA alternative splicing patterns.
Subject(s)
Autism Spectrum Disorder , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , RNA Splicing , RNA, Messenger/metabolism , Transcriptome , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/metabolism , Humans , RNA, Messenger/genetics , Sequence Analysis, RNAABSTRACT
Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.
Subject(s)
Alternative Splicing , Gene Regulatory Networks , Sequence Analysis, RNA/methods , Transcription Factors/metabolism , Animals , Cell Line , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , HEK293 Cells , Humans , Mice , Neurons/cytology , Neurons/metabolism , RNA, Messenger/geneticsABSTRACT
Alternative splicing is a key process underlying the evolution of increased proteomic and functional complexity and is especially prevalent in the mammalian nervous system. However, the factors and mechanisms governing nervous system-specific alternative splicing are not well understood. Through a genome-wide computational and expression profiling strategy, we have identified a tissue- and vertebrate-restricted Ser/Arg (SR) repeat splicing factor, the neural-specific SR-related protein of 100 kDa (nSR100). We show that nSR100 regulates an extensive network of brain-specific alternative exons enriched in genes that function in neural cell differentiation. nSR100 acts by increasing the levels of the neural/brain-enriched polypyrimidine tract binding protein and by interacting with its target transcripts. Disruption of nSR100 prevents neural cell differentiation in cell culture and in the developing zebrafish. Our results thus reveal a critical neural-specific alternative splicing regulator, the evolution of which has contributed to increased complexity in the vertebrate nervous system.