RNAseq in control and shQk KD mouse neural stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE103246 | GEO | 2017/12/08
SECONDARY ACCESSION(S): PRJNA400617
REPOSITORIES: GEO
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Project description:RNAseq in control and shQk KD mouse neural stem cells, and Qki5 CLIP in E14.5 mouse brain
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Project description:The mechanisms by which entire programs of gene regulation emerged during evolution are poorly understood. Neuronal microexons represent the most conserved class of alternative splicing in vertebrates and are critical for proper brain development and function. Here, we discover neural microexon programs in non-vertebrate species and trace their origin to bilaterian ancestors through the emergence of a previously uncharacterized ‘enhancer of microexons' (eMIC) protein domain. The eMIC domain originated as an alternative, neural-enriched splice isoform of the pan-eukaryotic Srrm2/SRm300 splicing factor gene, and subsequently became fixed in the vertebrate and neuronal-specific splicing regulator Srrm4/nSR100 and its paralog Srrm3. Remarkably, the eMIC domain is necessary and sufficient for microexon splicing, and functions by interacting with the earliest components required for exon recognition. The emergence of a novel domain with restricted expression in the nervous system thus resulted in the evolution of splicing programs that contributed to qualitatively expand neuronal molecular complexity in bilaterians.
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Project description:The human neural retina is enriched for alternative splicing, and it is estimated that more than 10% of variants associated with inherited retinal diseases (IRDs) alter splicing. Previous research mainly used short-read RNA-sequencing techniques to investigate retina-specific splicing and splicing factors. However, this technique provides limited information about transcript isoforms. To gain a deeper understanding of the human neural retina and its isoforms, we generated a proteogenomic atlas that combined PacBio long-read RNA-sequencing data with mass-spectrometry and whole-genome sequencing data from three healthy human neural retina samples. RNA-sequencing revealed that one-third of all transcripts were novel, and for IRD-associated genes, even 43% were novel. The most common novel elements of these transcripts were alternative poly(A) sites, exon elongation, and intron retention. Some novel elements affect the non-coding region but for more than 50% of the novel transcripts a novel open reading frame was predicted. Using proteomics, ten novel peptides confirmed novel isoforms in five genes. Additionally, we found novel isoforms of IMPDH1, an IRD-associated gene, with supporting peptide evidence. This study provides a comprehensive overview of the transcript and protein isoforms expressed in the healthy human neural retina. Moreover, it highlights the importance of studying tissue specific transcriptomes in greater detail to better understand tissue-specific regulation and to identify disease-causing variants.
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Project description:We sequenced mRNA from mouse E14.5 embryonic cortex to compare gene expression level and alternative splicing events between 2 control (WT) and 2 Qk cKO. A set of tissue specific splicing factors are thought to govern alternative splicing events during neural progenitors (NPC) to neuron transition by regulating neuron specific exons. Here we proposed one such a factor, RNA-binding protein Qki5, which is specifically expressed in neural stem cells. We performed mRNAseq analysis by using mRNAs obtained from developing cerebral cortices in Qk conditional knockout (cKO) mice. Expectedly, we found huge numbers of alternative splicing changes between control and conditional knock-out relative to that of transcripts level changes. Furthermore, DAVID and Meta-scape analysis revealed that affected spliced genes are involved in axon-development and microtubule-based process. Among these, Ninein protein coding mRNA is listed as a Qk protein dependent alternative splicing targets. Interestingly, this exon encodes very long poly-peptides (2,121 nt) and is known as a previously defined dynamic RNA switch during NPC-to-neuron transition. In addition, we validated that the regulation of this large exon is consistent with Qki5 dependent alternative exon inclusion mode obtained from our previous Qki5 HITS-CLIP analysis. Together Qki5 will add to a list factor of alternative splicing in NPC-to-neuron transition.
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Project description:Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as one important mechanism regulating cell fate decisions. Here we address the role of the evolutionary conserved splicing co-factor Barricade (Barc)/CUS2/Tat-SF1 in Drosophila neural stem cell (neuroblast) lineage formation. We show that Barc is required for the generation of neurons during Drosophila brain development by ensuring correct neural progenitor proliferation and differentiation. Barc associates with components of the U2 small nuclear ribonucleic proteins (snRNP), and its depletion causes alternative splicing in form of intron retention in a subset of genes. Using bioinformatics analysis and a cell culture based splicing assay, we found that Barc dependent introns share three major traits: they are short, GC rich and have weak 3’ splice sites. Our results show that Barc, together with the U2snRNP, plays an important role in regulating neural stem cell lineage progression during brain development and facilitates correct splicing of a subset of introns.
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Project description:RNAseq in control and shQk KD mouse neural stem cells
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Project description:We performed single cell transcriptomic profiling of induced human pluripotent stem cells (iPSCs)-derived type 2 alveolar epithelial cells (iAT2). iPSCs stably expressed CRISPRi (dCas9-KRAB) under the control of doxycyline. iAT2s were transduced with a lentivirus expressing gRNA targeting the transcriptional start site of DSP. Cells were treated with or without doxycyline to intiate CRISPRi-knockdown. Prior to capture, cells were labelled with hashing antibodies (HTO). Cells were captured for 10x Genomics Single cell capture (V3 protocol), and GEX and HTO libraries were sequenced. HTODemux function was used to demultiplex the samples. Knockdown of DSP caused cells to cluster separately from wild-type cells.
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Project description: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. A microarray platform to profile alternative splicing levels for 8714 cassette-type alternative exons across a diverse spectrum of mouse tissues.
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Project description:CC-671 has been identified as an inhibitor of Cdc2-like kinase 2 (CLK2) and TTK in direct enzyme assays. CLK2 is a member of the CLK family that phosphorylates serine- and arginine-rich (SR) proteins of the spliceosomal complex as part of a regulatory mechanism for control of pre-mRNA splicing. SR proteins are a family of small nuclear ribonucleoprotein particle (snRNP) splicing factors involved in constitutive and alternative splicing. Monitoring specific phospho-biomarkers of CLK2 demonstrated that CC-671 inhibited phosphorylation of CLK2 substrates in cancer cells with mean IC50 of 549 nM in the triple negative breast cancer (TNBC) line CAL51. In this study, RNA sequencing approach was used to quantify the impact of CC-671 treatment on gene transcription and global alternative splicing in CAL51 cells. Differential exon usage analysis demonstrated that CC-671 changed alternative splicing of many genes. In addition, different sets of genes are impacted by CC-671 at both the alternative splicing and mRNA expression. Genes impacted by alternative splicing shared a set of common pathways with genes altered by mRNA expression. This result indicates that CC-671 regulates transcription via both gene expression and alternative splicing mechanisms.
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Project description:We sequenced mRNA from hiPSC-MNs to compare gene expression level and alternative splicing events between 3 control and 3 siQKI.
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