Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE37933 | GEO | 2012/07/12
SECONDARY ACCESSION(S): PRJNA165651
REPOSITORIES: GEO
Similar Datasets
Project description:Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and non-neuronal cells. However, in non-neuronal cells, the splicing of 3M-bM-^@M-^Y-terminal introns within these genes is repressed by polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery including the nuclear pore-associated protein Tpr and components of the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context. PTBP1 siRNA, PTBP1+PTBP2 siRNA, or control siRNA
2012-07-12 | E-GEOD-37933 | biostudies-arrayexpress
Project description:Intron retention (IR) is an alternative splicing event where mRNAs containing unspliced introns are exported to the cytoplasm and then either translated, giving rise to new protein isoforms, or degraded via the nonsense-mediated decay pathway. However, unspliced introns are also seen in nuclear RNA. Some of these are spliced at a low rate but do eventually become fully spliced and their respective mRNAs exported, while others stay retained and are recognized by nuclear decay machineries. IR has been shown to be regulated during granulocyte and neuronal differentiation and in some cancers, and a subset of the nuclear retained introns, called detained introns, have been implicated in growth control pathways. Despite many findings on IR and its biological significance, it is still not clear whether an incompletely spliced intron observed is a true retained intron that is exported as an mRNA or is a product of slow splicing that remains in the nucleus. A better understanding is needed of the molecular events that reduce intron excision rate and determine the release of an RNA for export. We have found that some genes in mouse embryonic stem cells (mESCs) produce RNAs that are highly associated with the high molecular-weight nuclear pellet (chromatin), rather than the soluble nucleoplasm or cytoplasm. This chromatin-associated RNA is polyadenylated, usually contains one or more incompletely spliced introns, and is often extensively bound by polypyrimidine tract-binding protein 1 (PTBP1). We hypothesize that PTBP1 inhibits splicing of these introns, and this causes sequestration of the transcript on chromatin. We are studying the role of PTBP1 in inhibiting intron excision and anchoring RNAs in the nuclear and chromatin compartments of mESCs.
2020-11-18 | GSE159993 | GEO
Project description:We developed an extensive resource of RNAseq data to track mRNA maturation across subcellular locations in multiple cell types. Mouse embryonic stem cells, neuronal progenitor cells, and post-mitotic neurons were separated into chromatin-associated, soluble nucleoplasmic and cytoplasmic fractions, with RNA from each fraction isolated as both polyadenylated, total rRNA-depleted pools, and small RNA pools and sequenced. Genes exhibit disparate patterns of RNA enrichment between subcellular fractions, with some genes maintaining more polyadenylated RNA in the chromatin fraction than in the cytoplasm. These chromatin-associated poly(A)+ RNAs are often incompletely spliced and we devise a simple metric to identify introns whose excision is posttranscriptional. X-means clustering and deep learning methods identified intron groups with four defined regulatory behaviors, including complete cotranscriptional splicing, and complete retention in the cytoplasmic RNA. Two groups display intermediate levels of retention in the RNA in the nucleus but are fully spliced in the cytoplasm. These groups include previously described sets of retained introns, but extend to many new introns, including introns repressed by polypyrimidine track binding protein 1 (PTBP1). We find that the PTBP1 regulated Gabbr1 transcript is highly expressed in mESC without expression of Gabbr1 protein. The bulk of Gabbr1 RNA is incompletely spliced and remains sequestered on chromatin similar to certain long noncoding RNAs. Only after neuronal differentiation does Gabbr1 RNA become fully processed and released to the cytoplasm as mRNA. Thus, splicing repression and the chromatin anchoring of RNA provide a combined mechanism of posttranscriptional regulation over development. Our datasets provide a rich resource for analyzing many other aspects of mRNA maturation in subcellular locations and across development.
2020-11-18 | GSE159919 | GEO
Project description:We developed an extensive resource of RNAseq data to track mRNA maturation across subcellular locations in multiple cell types. Mouse embryonic stem cells, neuronal progenitor cells, and post-mitotic neurons were separated into chromatin-associated, soluble nucleoplasmic and cytoplasmic fractions, with RNA from each fraction isolated as both polyadenylated, total rRNA-depleted pools, and small RNA pools and sequenced. Genes exhibit disparate patterns of RNA enrichment between subcellular fractions, with some genes maintaining more polyadenylated RNA in the chromatin fraction than in the cytoplasm. These chromatin-associated poly(A)+ RNAs are often incompletely spliced and we devise a simple metric to identify introns whose excision is posttranscriptional. X-means clustering and deep learning methods identified intron groups with four defined regulatory behaviors, including complete cotranscriptional splicing, and complete retention in the cytoplasmic RNA. Two groups display intermediate levels of retention in the RNA in the nucleus but are fully spliced in the cytoplasm. These groups include previously described sets of retained introns, but extend to many new introns, including introns repressed by polypyrimidine track binding protein 1 (PTBP1). We find that the PTBP1 regulated Gabbr1 transcript is highly expressed in mESC without expression of Gabbr1 protein. The bulk of Gabbr1 RNA is incompletely spliced and remains sequestered on chromatin similar to certain long noncoding RNAs. Only after neuronal differentiation does Gabbr1 RNA become fully processed and released to the cytoplasm as mRNA. Thus, splicing repression and the chromatin anchoring of RNA provide a combined mechanism of posttranscriptional regulation over development. Our datasets provide a rich resource for analyzing many other aspects of mRNA maturation in subcellular locations and across development.
2020-11-18 | GSE159971 | GEO
Project description:We developed an extensive resource of RNAseq data to track mRNA maturation across subcellular locations in multiple cell types. Mouse embryonic stem cells, neuronal progenitor cells, and post-mitotic neurons were separated into chromatin-associated, soluble nucleoplasmic and cytoplasmic fractions, with RNA from each fraction isolated as both polyadenylated, total rRNA-depleted pools, and small RNA pools and sequenced. Genes exhibit disparate patterns of RNA enrichment between subcellular fractions, with some genes maintaining more polyadenylated RNA in the chromatin fraction than in the cytoplasm. These chromatin-associated poly(A)+ RNAs are often incompletely spliced and we devise a simple metric to identify introns whose excision is posttranscriptional. X-means clustering and deep learning methods identified intron groups with four defined regulatory behaviors, including complete cotranscriptional splicing, and complete retention in the cytoplasmic RNA. Two groups display intermediate levels of retention in the RNA in the nucleus but are fully spliced in the cytoplasm. These groups include previously described sets of retained introns, but extend to many new introns, including introns repressed by polypyrimidine track binding protein 1 (PTBP1). We find that the PTBP1 regulated Gabbr1 transcript is highly expressed in mESC without expression of Gabbr1 protein. The bulk of Gabbr1 RNA is incompletely spliced and remains sequestered on chromatin similar to certain long noncoding RNAs. Only after neuronal differentiation does Gabbr1 RNA become fully processed and released to the cytoplasm as mRNA. Thus, splicing repression and the chromatin anchoring of RNA provide a combined mechanism of posttranscriptional regulation over development. Our datasets provide a rich resource for analyzing many other aspects of mRNA maturation in subcellular locations and across development.
2020-11-18 | GSE159944 | GEO
Project description:We examined the role of PTBP1 in regulation of co-transcriptional splicing process by depleting this RNA-binding protein from embryonic stem cells using the auxin-inducible degron technology and analysing the total and chromatin-associated RNA fractions by RNA-seq. We also performed mNET-seq and ChIP-seq analyses using RNA polymerase II- and PTBP1-specific antibodies, respectively. Our data suggest that PTBP1 activates co-transcriptional splicing of hundreds of introns, a surprising effect given that PTBP1 is better known as a splicing repressor. Importantly, some co-transcriptionally activated introns fail to be spliced post-transcriptionally without PTBP1. In a striking example of this regulation, lasting retention of a PTBP1-dependent intron triggers nonsense-mediated decay of mRNAs encoding DNA methyltransferase DNMT3B, explaining their natural expression dynamics in development. Our further analyses suggest that this mechanism may protect differentiation-specific genes from aberrant methylation. We conclude that PTBP1-activated co-transcriptional splicing underlies biologically important decisions.
2023-01-08 | E-MTAB-11544 | biostudies-arrayexpress
Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. HB9-GFP mESCs were differentiated into mNPCs and mMNs. Poly-A RNA was isolated from isolated populations of mESCs, mNPCs, and mMNs for RNA-sequencing and splicing analyses.
2016-07-03 | E-GEOD-71079 | biostudies-arrayexpress
Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. 46C mESCs and mNPCs were cross-linked at 100 mJ/cm2. Cell pellets were collected and flash-frozen for iCLIP library preparation. Libraries were subjected to 100 single-end RNA-sequencing.
2016-07-03 | E-GEOD-71178 | biostudies-arrayexpress
Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. 46C mESCs were differentiated in mNPCs. The mNPCs were treated with 10 nM control, Ptbp1, Ptbp2, or Ptbp1 and Ptbp2 siRNAs for 48 hours. The knockdowns were performed using 2 independent sets of siRNAs. Poly-A RNA was isolated for RNA-sequencing and splicing analyses.
2016-07-03 | E-GEOD-70985 | biostudies-arrayexpress
Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. 46C mESCs were treated with 20 nM control, Ptbp1, Ptbp2, or Ptbp1 and Ptbp2 siRNAs for 72 hours. The knockdowns were performed using 2 independent sets of siRNAs, including one biological replicate. Poly-A RNA was isolated for RNA-sequencing and splicing analyses.
2016-07-03 | E-GEOD-71075 | biostudies-arrayexpress