Orchestrated intron retention regulates normal granulocyte differentiation
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: william ritchie
PROVIDER: E-GEOD-48307 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Using mRNA-seq, we determined intron retaining genes that were differentially regulated in FACS purified cells at three progressive stages of mouse granulopoiesis; CD34+Kit+Gr-1low promyelocytes, CD34-Kit-Gr-1mid myelocytes and CD34-Kit-Gr-1high granulocytes. We found that IR affects 86 genes, including those specific to granulocyte (Lyz2 and MMP8) and nuclear architecture (Lmnb1 and Lbr). IR was associated with the decrease in protein levels measured by mass spectrometry (P=0.0015, binomial test). Inhibition of NMD in granulocytes resulted in marked accumulation of 39/86 intron retaining mRNAs (P<0.05, RUV procedure with Holm-Bonferroni correction), indicating that IR triggers NMD to downregulate mRNA and protein expression. We used Affymetrix's Mouse Gene 1.0 ST array to determine accumulation of RNA-transcripts following inhibition of nonsense-mediated decay in primary mouse granulocytes using caffeine.
2013-06-27 | E-GEOD-48306 | biostudies-arrayexpress
Project description:Using mRNA-seq, we determined intron retaining genes that were differentially regulated in FACS purified cells at three progressive stages of mouse granulopoiesis; CD34+Kit+Gr-1low promyelocytes, CD34-Kit-Gr-1mid myelocytes and CD34-Kit-Gr-1high granulocytes. We found that IR affects 86 genes, including those specific to granulocyte (Lyz2 and MMP8) and nuclear architecture (Lmnb1 and Lbr). IR was associated with the decrease in protein levels measured by mass spectrometry (P=0.0015, binomial test). There was a significant overlap of IR between human and mouse (P=2.85E-22, hypergeometric test), showing that IR is conserved.Inhibition of NMD in granulocytes resulted in marked accumulation of 39/86 intron retaining mRNAs (P<0.05, RUV procedure with Holm-Bonferroni correction), indicating that IR triggers NMD to downregulate mRNA and protein expression. Sequencing of polyadenylated RNA from three types of myeloid cells (promyelocytes, myelocytes and granulocytes) using Illumina GAIIx
2013-06-27 | E-GEOD-48305 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
2013-06-27 | GSE48307 | GEO
Project description:Using mRNA-seq, we determined intron retaining genes that were differentially regulated in FACS purified cells at three progressive stages of mouse granulopoiesis; CD34+Kit+Gr-1low promyelocytes, CD34-Kit-Gr-1mid myelocytes and CD34-Kit-Gr-1high granulocytes. We found that IR affects 86 genes, including those specific to granulocyte (Lyz2 and MMP8) and nuclear architecture (Lmnb1 and Lbr). IR was associated with the decrease in protein levels measured by mass spectrometry (P=0.0015, binomial test). Inhibition of NMD in granulocytes resulted in marked accumulation of 39/86 intron retaining mRNAs (P<0.05, RUV procedure with Holm-Bonferroni correction), indicating that IR triggers NMD to downregulate mRNA and protein expression.
2013-06-27 | GSE48306 | GEO
Project description:Using mRNA-seq, we determined intron retaining genes that were differentially regulated in FACS purified cells at three progressive stages of mouse granulopoiesis; CD34+Kit+Gr-1low promyelocytes, CD34-Kit-Gr-1mid myelocytes and CD34-Kit-Gr-1high granulocytes. We found that IR affects 86 genes, including those specific to granulocyte (Lyz2 and MMP8) and nuclear architecture (Lmnb1 and Lbr). IR was associated with the decrease in protein levels measured by mass spectrometry (P=0.0015, binomial test). There was a significant overlap of IR between human and mouse (P=2.85E-22, hypergeometric test), showing that IR is conserved.Inhibition of NMD in granulocytes resulted in marked accumulation of 39/86 intron retaining mRNAs (P<0.05, RUV procedure with Holm-Bonferroni correction), indicating that IR triggers NMD to downregulate mRNA and protein expression.
2013-06-27 | GSE48305 | GEO
Project description:Orchestrated intron retention regulates normal granulocyte differentiation [RNA-Seq]
| PRJNA209629 | ENA
Project description:Inhibition of the nonsense mediated decay (NMD) mechanism in cells results in stabilization of transcripts carrying premature translation termination codons. A strategy referred to as gene indentification by NMD inhibition (GINI) has been proposed to identify genes carrying nonsense mutations (Noensie & Dietz, 2001). Genes containing frameshift mutations in colon cancer cell line have been identifying mutatnt genes using GINI, we have now further improved the strategy. In this approach, inhibition of NMD with emetine is complemented with inhibiting NMD by blocking the phosphorylation of the hUpf1 protein with caffeine. In addition, to enhance the GINI strategy, comparing mRNA level alterations produced by inhibiting transcription alone or inhbiiting transcription together with NMD following caffeine pretreatment were used for the efficient identification of false positives produced as a result of stress response to NMD inhibition. To demonstrate the improved efficiency of this approach, we analyzed colon cancer cell lines showing microstellite instability. Bi-allelic inactivating mutations were found in the FXR1, SEC1L1, NCOR1, BAT3, PHD14, ZNF294, C190ORF5 genes as well as genes coding for proteins with yet unknown functions. Experiment Overall Design: Using GINI2 as a method of identifying novel biallelic truncating mutations in MSI+ colon cancer cells, various treatment conditions were applied to cultured colon cancer cell lines LS180 (MSI+) and SW480 (MSI-) and gene expression profiling was measured using Affymetrix GeneChip U133 Plus 2 arrays.
2008-06-13 | E-GEOD-5486 | biostudies-arrayexpress
Project description:Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis.
| S-EPMC5392497 | biostudies-literature
Project description:We used a modification of GINI analysis to identify genes containing premature translation termination codons (PTC) generated by nonsense or frameshift mutations in prostate cancer cell lines. The analysis was performed in two steps. In the first step nonsense mediated mRNA decay (NMD) was inhibited in prostate cancer cell lines using incubation with medium containing caffeine for 4 hours. Gene expression analysis of caffeine treated or untreated cells after this step detects mRNA accumulation that takes place for genes containing PTC and as well as for genes that show induction of transciption due to stress caused by NMD inhibition. In the second step either both transcription and NMD or transcription only are blocked by incubating cell in a medium containing either both actinomycin D and caffeine or actinomacin D only for 4 hours. Gene expression analysis after this second step detects mRNA degradation for genes containing PTC as well as for genes that show induction of transciption due to stress caused by NMD inhibition. The efficiency of mRNA degradation for genes containing PTC during this step depends on whether NMD is inhibited or not. The efficiency of mRNA degradation for stress response genes does not depend on whether NMD is inhibited or not. Our modified GINI protocol allows efficient separation of genes containing PTC from the genes that show mRNA increases due to stress response to NMD inhibition. Experiment Overall Design: The LNCaP and 22Rv1 cells were seeded into four 60 mm tissue culture plates each. Caffeine (10 mM) was added to three plates and one plate was incubated without caffeine as a control. Following four hours? incubation, the medium was removed from one plate (with caffeine) and from the control plate and total RNA was prepared using TRIZOL reagent, according to manufacturer?s instructions, and used for an Affymetrix U133Plus2.0 oligonucleotide array hybridization. The caffeine-containing medium of the two remaining plates was removed, the cells were washed twice with phosphate-buffered saline and the medium with actinomycin D (2 mkg/ml) together with caffeine (10 mM) was added to one plate. Actinomycin D alone was added to the other plate. Following another four hours? incubation, the total RNA from both plates was prepared and used for an Affymetrix U133Plus2.0 oligonucleotide array analysis.
2009-07-12 | E-GEOD-16856 | biostudies-arrayexpress
Project description:BACKGROUND:In-depth study of the intron retention levels of transcripts provide insights on the mechanisms regulating pre-mRNA splicing efficiency. Additionally, detailed analysis of retained introns can link these introns to post-transcriptional regulation or identify aberrant splicing events in human diseases. RESULTS:We present IntEREst, Intron-Exon Retention Estimator, an R package that supports rigorous analysis of non-annotated intron retention events (in addition to the ones annotated by RefSeq or similar databases), and support intra-sample in addition to inter-sample comparisons. It accepts binary sequence alignment/map (.bam) files as input and determines genome-wide estimates of intron retention or exon-exon junction levels. Moreover, it includes functions for comparing subsets of user-defined introns (e.g. U12-type vs U2-type) and its plotting functions allow visualization of the distribution of the retention levels of the introns. Statistical methods are adapted from the DESeq2, edgeR and DEXSeq R packages to extract the significantly more or less retained introns. Analyses can be performed either sequentially (on single core) or in parallel (on multiple cores). We used IntEREst to investigate the U12- and U2-type intron retention in human and plant RNAseq dataset with defects in the U12-dependent spliceosome due to mutations in the ZRSR2 component of this spliceosome. Additionally, we compared the retained introns discovered by IntEREst with that of other methods and studies. CONCLUSION:IntEREst is an R package for Intron retention and exon-exon junction levels analysis of RNA-seq data. Both the human and plant analyses show that the U12-type introns are retained at higher level compared to the U2-type introns already in the control samples, but the retention is exacerbated in patient or plant samples carrying a mutated ZRSR2 gene. Intron retention events caused by ZRSR2 mutation that we discovered using IntEREst (DESeq2 based function) show considerable overlap with the retained introns discovered by other methods (e.g. IRFinder and edgeR based function of IntEREst). Our results indicate that increase in both the number of biological replicates and the depth of sequencing library promote the discovery of retained introns, but the effect of library size gradually decreases with more than 35 million reads mapped to the introns.
| S-EPMC5896110 | biostudies-literature