Effects of CRISPR/Cas9-mediated deletion of Safb1 and Safb2 genes on the mature miRNA transcriptome
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ABSTRACT:
PROVIDER: PRJNA592072 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10544939.fastq.gz | Fastqsanger.gz | |||
| SRR10544940.fastq.gz | Fastqsanger.gz | |||
| SRR10544941.fastq.gz | Fastqsanger.gz | |||
| SRR10544942.fastq.gz | Fastqsanger.gz | |||
| SRR10544943.fastq.gz | Fastqsanger.gz |
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Project description:MicroRNAs (miRNAs) are 20-25 nucleotides (nt) RNAs that are predicted to post-transcriptionally silence expression of >60% of all protein coding genes in mammals. As such, they form an additional layer of gene regulation that controls most if not all biological processes. MiRNAs are generated from primary transcripts containing single or multiple clustered stem-loop structures that are thought to be recognized and cleaved by the DGCR8/DROSHA Microprocessor complex as independent entities. Contrasting this view, we find that the primary miR-15a stem-loop within the bicistronic miR-15a-16-1 cluster is a poor Microprocessor substrate and is consequently not processed on its own, but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted primary miR-15 cleavage. Small RNA sequencing further revealed that deletion of Safb1 and 2 genes reduces not only miR-15a levels, but also affects other clustered miRNAs, among them miR-15b, miR-181b and miR-92a. We therefore postulate a model in which SAFB2 enables processing of suboptimal substrates in a subset of clustered pri-miRNA transcripts.
2019-11-28 | GSE141098 | GEO
Project description:The scaffold attachment factors SAFB1 and SAFB2 are paralogs, which are involved in cell cycle regulation, apoptosis, differentiation, and stress response. They have been shown to function as estrogen receptor co-repressors, and there is evidence for a role in breast tumorigenesis. To identify their endogenous target genes in MCF-7 breast cancer cells, we utilized gene expression array analysis, which was set up in a two-by-four design, with vehicle and estrogen treatment, and control, SAFB1, SAFB2, and SAFB1/SAFB2 siRNA as variables. Using custom chips containing 1.5 kb upstream regulatory region, we identified 541 SAFB1/SAFB2 binding sites in promoters of known genes, with significant enrichment on chromosome 1 and 6. Gene expression analysis revealed that the majority of target genes were induced in the absence of SAFB1 or SAFB2, and less were repressed. In contrast to SAFB2, which shared most of its target genes with SAFB1, SAFB1 had many unique target genes, most of them involved in regulation of the immune system. A subsequent analysis of the estrogen treatment group revealed that twelve percent of estrogen-regulated genes were dependent on SAFB1, with the majority being estrogen-repressed genes. These were primarily genes involved in apoptosis, such as BBC3, NEDD9, and OPG. Thus, this study confirms SAFB1/SAFB2âs primary role as co-repressors, and also uncovers a previously unknown role for SAFB1 in regulation of immune genes, and in estrogen-mediated repression of genes. To identify transcripts which were altered upon decrease of SAFB1 or SAFB2 expression we performed gene expression array analysis in MCF-7 cells. A secondary goal was to identify ERα-target genes for which estrogen regulation was dependent upon SAFB expression. We also added an experimental group in which we co-transfected siRNAs targeting both SAFB1 and SAFB2, which allowed us to address the question of interactions between the two paralogs. We therefore set up an experiment reflecting a 2x4 design, with a total of eight experimental groups.
2010-02-21 | E-GEOD-15548 | biostudies-arrayexpress
Project description:We used iCLIP to investigate the RNA interactions of the RNA binding protein SAFB1
2015-12-01 | GSE75418 | GEO
Project description:The scaffold attachment factors SAFB1 and SAFB2 are paralogs, which are involved in cell cycle regulation, apoptosis, differentiation, and stress response. They have been shown to function as estrogen receptor co-repressors, and there is evidence for a role in breast tumorigenesis. To identify their endogenous target genes in MCF-7 breast cancer cells, we utilized gene expression array analysis, which was set up in a two-by-four design, with vehicle and estrogen treatment, and control, SAFB1, SAFB2, and SAFB1/SAFB2 siRNA as variables. Using custom chips containing 1.5 kb upstream regulatory region, we identified 541 SAFB1/SAFB2 binding sites in promoters of known genes, with significant enrichment on chromosome 1 and 6. Gene expression analysis revealed that the majority of target genes were induced in the absence of SAFB1 or SAFB2, and less were repressed. In contrast to SAFB2, which shared most of its target genes with SAFB1, SAFB1 had many unique target genes, most of them involved in regulation of the immune system. A subsequent analysis of the estrogen treatment group revealed that twelve percent of estrogen-regulated genes were dependent on SAFB1, with the majority being estrogen-repressed genes. These were primarily genes involved in apoptosis, such as BBC3, NEDD9, and OPG. Thus, this study confirms SAFB1/SAFB2’s primary role as co-repressors, and also uncovers a previously unknown role for SAFB1 in regulation of immune genes, and in estrogen-mediated repression of genes.
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Project description:MicroRNAs are small non-coding RNAs that mediate post-transcriptional silencing of most mammalian genes. They are generated in a multi-step process initiated by the Microprocessor, a protein complex composed of DROSHA and DGCR8. Recent studies have described the phenomenon of “cluster assistance”, in which a prototypic primary miRNA hairpin can license the Microprocessor-mediated processing of a clustered suboptimal hairpin in cis. Mechanistic analyses led to the identification of two critical factors for this process, SAFB2 (scaffold attachment factor B2) and ERH (enhancer of rudimentary homolog), but it remains unclear to which extent these factors contribute to and are required for cluster assistance. Here, we have used a previously established dual fluorescence reporter system (doi: 10.1016/j.molcel.2020.05.011) to generate loss-of-function clones for SAFB2 and its closely related family member SAFB1 as well as for ERH.Our data indicate that loss of SAFB1/2 and of ERH results in overlapping, but not identical defects in primary miRNA biogenesis.
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Project description:Oxygen deprivation (hypoxia) is encountered in physiological conditions but also characterizes many pathological conditions including ischemia and cancer. In order to survive, the cells usually rely upon the activation of the Hypoxia Inducible Factors (HIF), a small family of heterodimeric transcriptional activators. However, adaption to hypoxia also involves, lesser-known, HIF-independent processes that affect chromatin remodelling and nuclear architecture. SAFB1/2 (Scaffold Attachment Factors B1 and 2) are integral components of the nuclear matrix of vertebrate cells and known to affect functions that include transcriptional regulation, RNA processing and response to DNA damage. Our work indicates that SAFB1/2 swiftly change their localization from the insoluble matrix fraction to more soluble nuclear fractions in a HIF-independent manner. To extend our study, we performed immunoprecipitation experiments with SAFB1 or SAFB2 from cells exposed to both normoxia and short-term hypoxia (2 hours). Mass spectrometry analysis of the immunoprecipitates revealed that short-term exposure of cells to low oxygen levels alters the protein-protein interactions of SAFB1/2 mainly with proteins implicated in mRNA maturation and transcriptional control. Our data suggest that SAFB1/2 proteins participate in molecular mechanisms immediately responsive to hypoxia that entail their rapid mobilization from the nuclear matrix and their altered association with constituents of splicing and transcriptional machinery.
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Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library was performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
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Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
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Project description:NFYC-AS1 is an overlapping antisense RNA transcribed head-to-head to NFYC sense gene, encoding for the subunit C of NF-Y transcription factor, which is known as master regulator of cell cycle and proliferation in normal and tumor cells. Here we performed NFYC-AS1 silencing in lung squamous carcinoma H520 cells by Gapmer antisense oligonucleotides and CRISPR/Cas9 TSS deletion. Afterwards, we performed differentially expressed analysis and gene set enrichement analysis to investigate on NFYC-AS1 function and mechanism of action.
2024-03-08 | GSE240468 | GEO
Project description:Disruption of miRNA Sequence by TALENs and CRISPR/Cas9 Induces Varied Length of miRNA Production [CRISPR/Cas9]
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