- http://www.crg.eu/en/group-members/panagiotis-papasaikas
Additional file 1. Supplementary figures and corresponding legends.
Background Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming,... more
Background Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators...
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In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for cell reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood.... more
In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for cell reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we have studied the dynamics of alternative splicing (AS) changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells. These changes, generally uncoupled from transcriptional regulation, significantly overlapped with splicing programs reported during reprogramming of mouse embryonic fibroblasts (MEFs). Correlation between gene expression of potential regulators and specific clusters of AS changes enabled the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of MEFs reprogramming. These RNA-binding proteins control partially overlapping programs of splicing regulation affecting genes involved in developmental and morphogenetic processes. Our resu...
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Mechanical forces shape cell fate decisions during development and regeneration in many systems. Epithelial lumen volume changes, for example, generate mechanical forces that can be perceived by the surrounding tissue and integrated into... more
Mechanical forces shape cell fate decisions during development and regeneration in many systems. Epithelial lumen volume changes, for example, generate mechanical forces that can be perceived by the surrounding tissue and integrated into cell fate decisions. Similar behavior occurs in regenerating Hydra tissue spheroids, where periodic osmotically driven inflation and deflation cycles generate mechanical stimuli in the form of tissue stretching. Using this model, we investigate how such mechanical input guides the de novo formation of differentiated body parts. We show that the expression of the organizer-defining factor Wnt3 functions as a quantitative readout of cellular stretching and, when supplied externally, enables successful regeneration without mechanical stimulation. This finding represents a previously undescribed cellular mechanism for converting mechanical stimuli to a biochemical signaling readout and guiding cell fate transitions. It also elucidates the role of mechan...
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ABSTRACTMouse embryonic stem cells (mESCs) give rise to embryonic but not extraembryonic endoderm fates. Here, we identify the mechanism of this lineage barrier and report that the histone deacetylase Hdac3 and the corepressor Dax1... more
ABSTRACTMouse embryonic stem cells (mESCs) give rise to embryonic but not extraembryonic endoderm fates. Here, we identify the mechanism of this lineage barrier and report that the histone deacetylase Hdac3 and the corepressor Dax1 cooperatively restrict transdifferentiation of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor (TF) Gata6. This restriction is opposed by the pluripotency TFs Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency, and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC TF network.
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SummaryHow closely human organoids recapitulate cell-type diversity and cell-type maturation of their target organs is not well understood. We developed human retinal organoids with multiple nuclear and synaptic layers. We sequenced the... more
SummaryHow closely human organoids recapitulate cell-type diversity and cell-type maturation of their target organs is not well understood. We developed human retinal organoids with multiple nuclear and synaptic layers. We sequenced the RNA of 158,844 single cells from these organoids at six developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable ‘developed’ state at a rate similar to human retina development in vivo and the transcriptomes of organoid cell types converged towards the transcriptomes of adult peripheral retinal cell types. The expression of disease-associated genes was significantly cell-type specific in adult retina and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanis...
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Research Interests: Chemistry, Biology, Cell Biology, Stem Cell, Medicine, and 4 moreMultidisciplinary, Nature, Cell, and Organoid
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The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we... more
The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role ...
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We present a computational framework tailored for the modeling of the complex, dynamic relationships that are encountered in splicing regulation. The starting point is whole-genome transcriptomic data from high-throughput array or... more
We present a computational framework tailored for the modeling of the complex, dynamic relationships that are encountered in splicing regulation. The starting point is whole-genome transcriptomic data from high-throughput array or sequencing methods that are used to quantify gene expression and alternative splicing across multiple contexts. This information is used as input for state of the art methods for Graphical Model Selection in order to recover the structure of a composite network that simultaneously models exon co-regulation and their cognate regulators. Community structure detection and social network analysis methods are used to identify distinct modules and key actors within the network. As a proof of concept for our framework we studied the splicing regulatory network for Drosophila development using the publicly available modENCODE data. The final model offers a comprehensive view of the splicing circuitry that underlies fly development. Identified modules are associate...
Research Interests: Computational Biology, Biology, Computational Biophysics, Development, Medicine, and 15 moreBiological Sciences, Biological Networks, Alternative splicing, Genome, Gene Regulatory Networks, Regulatory Network, Module, Regulatory networks, Alternative Splicing, Regulator, mRNA processing, Graphical Model, Exon, Gene Expression Regulation, and Medical and Health Sciences
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Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of... more
Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and c...
Research Interests: Principal Component Analysis, Biology, Apoptosis, Medicine, Cell and Molecular Biology, and 13 moreBiological Sciences, Humans, Alternative splicing, Gene Regulatory Networks, Introns, HeLa cells, Alternative Splicing, Cell Proliferation, Ribonucleoproteins, Protein Binding, binding sites, Spliceosome, and Medical and Health Sciences
Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of... more
Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of endogenous transcripts in mammalian cells, we identified 200 genes whose knockdown modulates the ratio between Fas/CD95 isoforms. These include classical splicing regulators; core spliceosome components; and factors implicated in transcription and chromatin remodeling, RNA transport, intracellular signaling, and metabolic control. Coherent effects of genes involved in iron homeostasis and pharmacological modulation of iron levels revealed a link between intracellular iron and Fas/CD95 exon 6 inclusion. A splicing regulatory network linked iron levels with reduced activity of the Zinc-finger-containing splicing regulator SRSF7, and in vivo and in vitro assays revealed that iron inhibits SRSF7 RNA binding. Our results uncover numerous links between cellula...
Research Interests: Biology, Cell Biology, Apoptosis, Medicine, Biological Sciences, and 15 moreHumans, Alternative splicing, Iron, Genome, Gene Regulatory Networks, Introns, Homeostasis, HeLa cells, Alternative Splicing, Cell Proliferation, Exon, Deferoxamine, Mitochondrial Proteins, binding sites, and Medical and Health Sciences
Research Interests: Computer Science, Algorithms, Computational Biology, Biology, Medicine, and 15 moreBiological Sciences, Humans, Markov chains, Mathematical Sciences, Animals, hidden Markov model, BMC Bioinformatics, Cross Validation, G protein, Amino Acid Sequence, Experimental Data, Ligands, Molecular Sequence Data, BMC, and binding sites
Flexibility in regulating RNA splicing can generate diverse phenotypic differences among equivalent organs across vertebrates.
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Research Interests: Computer Science, Artificial Intelligence, Chemistry, Computational Biology, Biology, and 15 moreForecasting, Medicine, Biological Sciences, Environmental Sciences, Humans, Markov chains, Drug Design, hidden Markov model, Prescription drugs, CHEMICAL SCIENCES, Multiple Sequence Alignment, Genome sequence, Internet, Probabilistic Approach, and Drug prescriptions
Research Interests: Computer Science, Computational Biology, Biology, Medicine, The Internet, and 14 moreBiological Sciences, Software, Environmental Sciences, G protein-coupled receptors, Nucleic Acids, Computer User Interface Design, Protein Sequence Analysis, Data Flow Diagram, Internet services, Reproducibility of Results, Cell Surface Markers, Internet, Web Interface, and G Protein Coupled Receptors
Research Interests: Genetics, Biology, Dopamine, Haplotypes, Female, and 15 moreGenetic Association Studies, Alternative splicing, American, Genetic determinism, Clinical Sciences, Genotype, Dopamine Transporter, Catecholamine, Alternative Splicing, Genetic Association, Base Sequence, Genetic Mapping, Exon, alleles, and Gene frequency
Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via... more
Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA’s 5′ splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5′ and 3′ regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5′SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with ...
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The spliceosome, one of the most complex machineries of eukaryotic cells, removes intronic sequences from primary transcripts to generate functional messenger and long noncoding RNAs (lncRNA). Genetic, biochemical, and structural data... more
The spliceosome, one of the most complex machineries of eukaryotic cells, removes intronic sequences from primary transcripts to generate functional messenger and long noncoding RNAs (lncRNA). Genetic, biochemical, and structural data reveal that the spliceosome is an RNA-based enzyme. Striking mechanistic and structural similarities strongly argue that pre-mRNA introns originated from self-catalytic group II ribozymes. However, in the spliceosome, protein components organize and activate the catalytic-site RNAs, and recognize and pair together splice sites at intron boundaries. The spliceosome is a dynamic, reversible, and flexible machine that chaperones small nuclear (sn) RNAs and a variety of pre-mRNA sequences into conformations that enable intron removal. This malleability likely contributes to the regulation of alternative splicing, a prevalent process contributing to cell differentiation, homeostasis, and disease.
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We present a computational framework tailored for the modeling of the complex, dynamic relationships that are encountered in splicing regulation. The starting point is whole-genome transcriptomic data from high-throughput array or... more
We present a computational framework tailored for the modeling of the complex, dynamic relationships that are encountered in splicing regulation. The starting point is whole-genome transcriptomic data from high-throughput array or sequencing methods that are used to quantify gene expression and alternative splicing across multiple contexts. This information is used as input for state of the art methods for Graphical Model Selection in order to recover the structure of a composite network that simultaneously models exon co-regulation and their cognate regulators. Community structure detection and social network analysis methods are used to identify distinct modules and key actors within the network. As a proof of concept for our framework we studied the splicing regulatory network for Drosophila development using the publicly available modENCODE data. The final model offers a comprehensive view of the splicing circuitry that underlies fly development. Identified modules are associated with major developmental hallmarks including maternally loaded RNAs, onset of zygotic gene expression, transitions between life stages and sex differentiation. Within-module key actors include well-known developmental-specific splicing regulators from the literature while additional factors previously unassociated with developmental-specific splicing are also highlighted. Finally we analyze an extensive battery of Splicing Factor knock-down transcriptome data and demonstrate that our approach captures true regulatory relationships.